Method for the alteration of plants using cle polypeptides/peptides

ABSTRACT

The present invention relates to altering the biomass and/or structure of a plant, in order to maximise its potential as a source of feedstock or increase its potential as a feedstock for the paper industry. CLE41 and/or CLE42 are used to manipulate growth and structure of the vascular tissue of the plant. The present invention also provides plants in which the levels of CLE41 and/or CLE42 are increased compared to those of a native plant grown under identical conditions, and parts of such plants. Also provided are methods for using such plants or plant parts in the production of plant derived products such as paper or biofuels.

FIELD OF THE INVENTION

The present invention relates to manipulating the growth and/orstructure of a plant through modulation of the amount of CLE41 and/orCLE42 expressed in the plant, and additionally or alternatively,modulating the amount of PXY in the plant. Manipulating the growthand/or structure of a plant can be used to alter the mechanicalproperties of a plant or plant derived product, or to maximise itspotential for the production of plant derived products such as biofuelsand paper.

BACKGROUND

In multi-cellular organisms, cells must communicate with each other inorder for growth and development to occur in an ordered manner. Inanimals, it has long been known that polypeptides act as signallingmolecules in mediating communication between cells, a common examplebeing insulin in humans. These signalling molecules are responsible forinitiating many cellular processes, typically by binding to a receptorat the cell surface, which in turn transmits a message to inside thecell via downstream signalling proteins such as membrane associatedprotein kinases (MAPK), tyrosine phosphatases and Ras proteins. In thecell, the cell signalling pathway end-point is usually a transcriptionfactor target, which mediates a change in gene expression in the cell,thus causing a change in the growth and/or development of the cell inresponse to the initial extracellular signal.

In plants, it is also known that cell signalling occurs, and this wasthought to be mediated by plant hormones such as auxin and cytokinin.More recently, the discovery of systemin has shown that polypeptidesalso play a role in cell-signalling in plants. One of the largestfamilies of signalling polypeptides identified in plants is the Clavata3(clv3)/Endosperm Surrounding Region (ESR)-related (CLE) family. Theseproteins are the most highly characterised family of small polypeptidesin plants. The Arabidopsis thaliana genome contains 32 CLE genes. Clv3is the best characterised CLE family member which acts together with areceptor kinase (CLAVATA 1) to play a role in regulating theproliferation of cells in the shoot (apical) meristem. At present,however, most of the CLE family remain functionally undefined.

The CLE gene family has been shown to be present in a variety of otherplant species (Jun et al Cell. Mol. Life. Sci. 65 743-755 (2008) andFrickey et al BMC Plant Biology 2008, 8:1 10.1186/1471-2229-8-1)including rice, maize, tomato and alfalfa.

The polypeptides encoded by the CLE genes share common characteristics.They are less than 15 kDa in mass and comprise a short stretch ofhydrophobic amino acids at the amino terminus which serves to target thepolypeptide to the secretory pathway. This conserved stretch of 14 aminoacids is known as the CLE domain (Jun et al supra).

Higher plants show post-embyronic development at shoot and root tips,which are known as the apical meristems. Stem cells at these meristemsproduce cells which differentiate to become flower, leaf, stem or rootcells. A loss-of-function mutant resulting in an excess of stem cells atthe apical meristem suggests that Clv3 plays a role in regulation ofgrowth and/or differentiation at the growing tip. Over expression ofCLV3 results in loss of apical stem cells, thus post-embryonic aboveground parts of the plant are lost. The signalling pathway which CLV3regulates has been elucidated and is described in Jun et al (supra).This pathway is thought to be conserved amongst other plants species.

Shiu and Bleecker suggest that the CLE family is likely to coordinatewith a group of plant receptors known as the leucine-rich-repeatreceptor-like (LLR-RLK) kinases (PNAS 98 10763-10768 (2001)).

U.S. Pat. No. 7,179,963 describes a maize clv3-like nucleotide sequence,and its use in modulating plant development and differentiation. U.S.Pat. No. 7,335,760 discloses nucleic acid sequences for use ingenetically modifying a plant to increase plant yield and the mass ofthe plant, for example for biofuel production.

Other CLE family members have been shown to inhibit celldifferentiation. For example, Frickey et al (supra) have looked at theCLE family and suggested that CLE family members CLE41 and/or 42 mayplay a role in vascular development. Ito et al (Science Vol 313 842-845(2006)) show that dodecapeptides are important in preventing vascularcell differentiation.

In contrast, however, Strabala et al (Plant Physiology vol. 1401331-1344 (2006)) show that CLE41 and/or 42 are genuine expressedmembers of the CLE family. Although general over-expression of CLE42throughout the plant results in a dwarf phenotype, Strabala et al reportthat CLE42 is likely to be a functionally redundant molecule.

The source of biomass in plants is their woody tissue, derived from thevascular meristems of the plant such as the cambium and procambium,which divide to form the phloem and xylem cells of the vascular tissuewithin the plant stems and roots. The cambium and procambium(collectively known as the vascular meristems) are growth zones whichenable the plant to grow laterally, thus generating the majority ofbiomass. Enhancing lateral growth by genetically altering the rates ofprocambial or cambial cell division may lead to an increase in the plantbiomass. This would provide an additional source of biomass for variousindustries dependent upon plant derived products, such as the biofuel orpaper industries.

Increasing the yield of biomass of plants, for example for paper andfuel production has previously been done by breeding programs, but inrecent years there is interest in the use of genetic manipulation orplant modification for such purposes.

The division of cells to form the vascular tissue is a highly orderedprocess. Prominent polarity of cells destined to become either phloemcells or xylem cells is observed, the latter eventually forming thewoody tissue of the plant. Xylem is principally water transportingtissue of the plant, and together with phloem, forms a vascular networkfor the plant. The cells of the xylem which are principally responsiblefor carrying water are the tracheary elements, of which there are twotypes—tracheids and vessels.

However, whilst there has been much investigation into the regulation ofgrowth at the apical meristems, there is less understanding of thegrowth of the vascular tissue. Fisher et al (Current Biology 171061-1066 (2007)) report a loss of function mutant in which the spatialorganisation of the vascular tissue is lost and the xylem and phloemcells are interspersed. The mutant is in a gene named PXY, which encodesa receptor-like kinase.

Tracheary elements (TEs) are cells in the xylem that are highlyspecialized for transporting water and solutes up the plant. They areproduced from xylem cells by a process which involves specification,enlargement, patterned cell wall deposition, programmed cell death andcell wall removal. This results in adjacent TEs being joined together toform a continuous network for water transport.

Jun et al (supra) disclose that the CLE domain of CLE41 is identical toTracheal Element Differentiation Inhibitory Factor (TDIF), which hasbeen shown to inhibit cell differentiation, and CLE42 differs by onlyone amino acid from the TDIF sequence. When exogenously applied to cellcultures, synthetic CLE41 and CLE42 suppressed the formation oftracheary element cells from the xylem (Ito et al, supra).

There remains a need for identification of genetic elements, themanipulation of which can be used to alter the growth and/or structureof the plant.

BRIEF SUMMARY OF THE INVENTION

In a first aspect the invention provides the use of a polypeptideselected from the group consisting of:

-   -   i) a CLE41 polypeptide;    -   ii) a CLE42 polypeptide;    -   iii) a polypeptide comprising an amino acid sequence that is at        least 70% identical to the amino acid sequence of amino acids        124 to 137 of the consensus sequence of FIG. 10 (SEQ ID NO. 12);    -   iv) a polypeptide comprising an amino acid sequence that is at        least 70% identical to the amino acid sequence of CLE41 of FIG.        13A (SEQ ID NO. 21) or CLE42 of FIG. 14A (SEQ ID NO. 23);    -   v) a polypeptide encoded by a nucleic acid molecule that is at        least 70% identical to the nucleotide sequence of CLE41 of FIG.        13B (SEQ ID NO. 22) or CLE42 of FIG. 14B (SEQ ID NO. 24);        in the manipulation of plant growth and/or structure.

In a second aspect the invention provides the use of a nucleic acidmolecule selected from the group consisting of:

-   -   i) a nucleic acid molecule that encodes a CLE41 polypeptide;    -   ii) a nucleic acid molecule that encodes a CLE42 polypeptide;    -   iii) a nucleic acid molecule that encodes a polypeptide        comprising an amino acid sequence that is at least 70% identical        to the amino acid sequence of amino acids 124 to 137 of the        consensus sequence of FIG. 10 (SEQ ID NO. 12);    -   iv) a nucleic acid molecule which is at least 70% identical to        the nucleotide sequence of CLE41 of FIG. 13B (SEQ ID NO. 22) or        CLE42 of FIG. 14B (SEQ ID NO. 24);    -   v) a nucleic acid molecule that hybridizes under stringent        conditions to the nucleotide sequence i) or ii)        in the manipulation of plant growth and/or structure.

Preferably, the use of the first or second aspect is use of thepolypeptide or nucleic acid in combination with a nucleic acid moleculeselected from the group consisting of:

-   -   i) a nucleic acid molecule that encodes a CLE41 receptor;    -   ii) a nucleic acid molecule that encodes a CLE42 receptor;    -   iii) a nucleic acid molecule that encodes a polypeptide        comprising an amino acid sequence that is at least 70% identical        to the consensus sequence of FIG. 12 (SEQ ID NO. 20), or a        functional equivalent thereof;    -   iv) a nucleic acid molecule that is at least 70% identical to        the nucleotide sequence of FIG. 15B (SEQ ID NO. 26) or 15C (SEQ        ID NO. 27);    -   v) a nucleic acid molecule that is at least 70% identical to a        nucleic acid molecule of i) or ii);    -   vi) a nucleic acid molecule that hybridizes under stringent        conditions to the nucleotide sequence i) or ii).

Preferably, the use of the first or second aspect is use of thepolypeptide or nucleic acid in combination with a polypeptide selectedfrom the group consisting of:

-   -   i) a CLE41 receptor;    -   ii) a CLE42 receptor;    -   iii) a polypeptide comprising an amino acid sequence that is at        least 70% identical to the consensus sequence of FIG. 12 (SEQ ID        NO. 20), or a functional equivalent thereof    -   iv) a polypeptide comprising an amino acid sequence that is at        least 70% identical to the PXY sequence of FIG. 15A (SEQ ID NO.        25);    -   v) a polypeptide sequence comprising an amino acid sequence        which is at least 70% identical to a sequence encoding i) or        ii);    -   vi) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule that is at least 70% identical to the PXY        nucleotide sequence of FIG. 15B (SEQ ID NO. 26) or 15C (SEQ ID        NO. 27).

Preferably said CLE41 or CLE42 receptor is PXY or a functionalequivalent thereof.

Preferably said manipulation of the plant growth and/or structure is anincrease or decrease in the amount of growth and/or division of theprocambial and/or cambial cells in a plant, specifically the number ofcells generated. More specifically, it is an increase or decrease in therate of division of such cells. Thus, the manipulation of growth and/orstructure can be said to be an increase or decrease in the secondarygrowth of the plant, and/or an increase or decrease in the degree oforganisation of the secondary structure, at the cellular level. Bysecondary growth is preferably meant the woody tissue of a plant, or thevascular or interfasicular tissue. Preferably, where there is anincrease in the number of procambial and/or cambial cells, these cellsdifferentiate into xylem and/or phloem cells, preferably the former.

In a further aspect, the present invention provides a method ofmanipulating the growth and/or structure of a plant, comprisingmodulating the level of CLE41 and/or CLE42 or a functional equivalentthereof, in the plant.

Preferably the levels of CLE41 and/or CLE42 are modulated by introducinginto a cell of the plant:

-   -   i) a CLE41 polypeptide;    -   ii) a CLE42 polypeptide;    -   iii) a polypeptide comprising an amino acid sequence that is at        least 70% identical to the amino acid sequence of amino acids        124 to 137 of the consensus sequence of FIG. 10 (SEQ ID NO. 12);    -   iv) a polypeptide comprising an amino acid sequence that is at        least 70% identical to the amino acid sequence of CLE41 of FIG.        13A (SEQ ID NO. 21) or CLE42 of FIG. 14A (SEQ ID NO. 23);    -   v) a polypeptide encoded by a nucleic acid molecule that is at        least 70% identical to the nucleotide sequence of CLE41 of FIG.        13B (SEQ ID NO. 22) or CLE42 of FIG. 14B (SEQ ID NO. 24).

Alternatively the levels of CLE41 and/or CLE42 are modulated byintroducing into a cell of the plant:

-   -   i) a nucleic acid molecule that encodes a CLE41 polypeptide;    -   ii) a nucleic acid molecule that encodes a CLE42 polypeptide;    -   iii) a nucleic acid molecule that encodes a polypeptide        comprising an amino acid sequence that is at least 70% identical        to the amino acid sequence of amino acids 124 to 137 of the        consensus sequence of FIG. 10 (SEQ ID NO. 12);    -   iv) a nucleic acid molecule which is at least 70% identical to        the nucleotide sequence of CLE41 of FIG. 13B (SEQ ID NO. 22) or        CLE42 of FIG. 14B (SEQ ID NO. 24);    -   v) a nucleic acid molecule that hybridizes under stringent        conditions to the nucleotide sequence i) or ii).

Preferably, the levels of levels of CLE41 and/or CLE42 or a functionalequivalent thereof are upregulated.

Optionally, the method further comprises introducing into a cell of theplant:

-   -   i) a nucleic acid molecule that encodes a CLE41 receptor;    -   ii) a nucleic acid molecule that encodes a CLE42 receptor;    -   iii) a nucleic acid molecule that encodes a polypeptide        comprising an amino acid sequence that is at least 70% identical        to the consensus sequence of FIG. 12 (SEQ ID NO. 20), or a        functional equivalent thereof;    -   iv) a nucleic acid molecule that is at least 70% identical to        the nucleotide sequence of FIG. 15B (SEQ ID NO. 26) or 15C (SEQ        ID NO. 27);    -   v) a nucleic acid molecule that is at least 70% identical to a        nucleic acid molecule of i) or ii);    -   vi) a nucleic acid molecule that hybridizes under stringent        conditions to the nucleotide sequence i) or ii).

Alternatively, the method further comprises introducing into cell of aplant:

-   -   i) a CLE41 receptor;    -   ii) a CLE42 receptor;    -   iii) a polypeptide comprising an amino acid sequence that is at        least 70% identical to the consensus sequence of FIG. 12 (SEQ ID        NO. 20), or a functional equivalent thereof    -   iv) a polypeptide comprising an amino acid sequence that is at        least 70% identical to the PXY sequence of FIG. 15A (SEQ ID NO.        25);    -   v) a polypeptide sequence comprising an amino acid sequence        which is at least 70% identical to a sequence encoding i) or        ii);    -   vi) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule that is at least 70% identical to the PXY        nucleotide sequence of FIG. 15B (SEQ ID NO. 26) or 15C (SEQ ID        NO. 27).

Preferably said CLE41 and/or CLE42 receptor is PXY or a functionalequivalent thereof.

In aspects where the levels of two or more of CLE41, CLE42 and PXY areto be manipulated in a plant, this may be achieved by:

-   -   (i) manipulating the levels of CLE41 and/or CLE42 as        hereinbefore described, in a first plant;    -   (ii) manipulating the levels of a CLE41 and/or CLE42 receptor as        hereinbefore described, in a second plant;    -   (iii) crossing said first and second plants to obtain a plant in        which the levels of CLE41 and/or CLE42 and said receptor are        manipulated. Also provided in the present invention is the plant        produced by the crossing of the first and second plants, and        progeny thereof which express the non-native nucleotide and/or        polypeptide sequences.

In a further aspect, the present invention provides a plant cellmanipulated to express:

-   -   i) a CLE41 polypeptide;    -   ii) a CLE42 polypeptide;    -   iii) a polypeptide comprising an amino acid sequence that is at        least 70% identical to the amino acid sequence of amino acids        124 to 137 of the consensus sequence of FIG. 10 (SEQ ID NO. 12);    -   iv) a polypeptide comprising an amino acid sequence that is at        least 70% identical to the amino acid sequence of CLE41 of FIG.        13A (SEQ ID NO. 21) or CLE42 of FIG. 14A (SEQ ID NO. 23);    -   v) a polypeptide encoded by a nucleic acid molecule that is at        least 70% identical to the nucleotide sequence of CLE41 of FIG.        13B (SEQ ID NO. 22) or CLE42 of FIG. 14B (SEQ ID NO. 24);        optionally in combination with expression of a receptor for        CLE41 and/or CLE42.

In a further aspect, the present invention provides a plant cellmanipulated to express

-   -   i) a nucleic acid molecule that encodes a CLE41 polypeptide;    -   ii) a nucleic acid molecule that encodes a CLE42 polypeptide;    -   iii) a nucleic acid molecule that encodes a polypeptide        comprising an amino acid sequence that is at least 70% identical        to the amino acid sequence of amino acids 124 to 137 of the        consensus sequence of FIG. 10 (SEQ ID NO. 12);    -   iv) a nucleic acid molecule which is at least 70% identical to        the nucleotide sequence of CLE41 of FIG. 13B (SEQ ID NO. 22) or        CLE42 of FIG. 14B (SEQ ID NO. 24);    -   v) a nucleic acid molecule that hybridizes under stringent        conditions to the nucleotide sequence i) or ii).

Preferably, said plant cell is further manipulated to express a nucleicacid molecule selected from the group consisting of:

-   -   i) a nucleic acid molecule that encodes a CLE41 receptor;    -   ii) a nucleic acid molecule that encodes a CLE42 receptor;    -   iii) a nucleic acid molecule that encodes a polypeptide        comprising an amino acid sequence that is at least 70% identical        to the consensus sequence of FIG. 12 (SEQ ID NO. 20), or a        functional equivalent thereof;    -   iv) a nucleic acid molecule that is at least 70% identical to        the nucleotide sequence of FIG. 15B (SEQ ID NO. 26) or 15C (SEQ        ID NO. 27);    -   v) a nucleic acid molecule that is at least 70% identical to a        nucleic acid molecule of i) or ii);    -   vi) a nucleic acid molecule that hybridizes under stringent        conditions to the nucleotide sequence i) or ii).

Alternatively, said plant cell is further manipulated to express apolypeptide selected from the group consisting of:

-   -   i) a CLE41 receptor;    -   ii) a CLE42 receptor;    -   iii) a polypeptide comprising an amino acid sequence that is at        least 70% identical to the consensus sequence of FIG. 12 (SEQ ID        NO. 20), or a functional equivalent thereof    -   iv) a polypeptide comprising an amino acid sequence that is at        least 70% identical to the PXY sequence of FIG. 15A (SEQ ID NO.        25);    -   v) a polypeptide sequence comprising an amino acid sequence        which is at least 70% identical to a sequence encoding i) or        ii);    -   vi) a polypeptide comprising an amino acid sequence encoded by a        nucleic acid molecule that is at least 70% identical to the PXY        nucleotide sequence of FIG. 15B (SEQ ID NO. 26) or 15C (SEQ ID        NO. 27).

Preferably said CLE41 and/or CLE42 receptor is PXY or a functionalequivalent thereof.

In a further aspect, there is provided a nucleic acid molecule encodinga functional equivalent of PXY, preferably derived from Arabidopsisthaliana, poplar or rice, and more preferably encoding the amino acidsequence of the consensus sequence of FIG. 12 (SEQ ID NO. 20). Alsoprovided is a polypeptide sequence encoding a functional equivalent ofPXY, preferably derived from Arabidopsis thaliana, poplar or rice, andpreferably comprising an amino acid sequence of the consensus sequenceof FIG. 12 (SEQ ID NO. 20). Preferably, the amino acid sequencecomprises the sequence of pttPXY, PXYL-1, PXYL-2 or Os02g02140.1, orOs03g05140.1 of FIG. 12 ((SEQ ID NOs. 16, 18, 19, 13 and 14respectively). Also included are sequences having 70% sequence identityor sequence homology thereto.

It is apparent that the levels of CLE41, CLE42 and or a receptorthereof, such as PXY, in each of the aspects of the present inventionmay be manipulated by altering the expression of native CLE41, CLE42 andor a receptor thereof within the plant cell. This may be achieved byplacing the native nucleotide sequence under the control of a nucleotidesequence which modifies expression of a native gene to allow modifyexpression thereof. The nucleotide sequence may be a regulatorysequence, as defined herein, or may encode a regulatory protein, such asa transcription factor, or may encode a DNA or RNA antisense sequence.As such, the nucleotide sequence or its expression product can modifyexpression, amount and/or activity of a native gene/polypeptide. Methodsof function of such regulatory proteins, expression products andantisense will be known to persons skilled in the art.

In a yet further aspect, the present invention provides a plantcomprising a cell according to the invention. Also provided are progenyof the plants of the invention.

In a further aspect, there is provided the use of a cell or plant of theinvention in the production of a plant-derived product. A plant-derivedproduct may include biomass, fibres, forage, biocomposites, biopolymers,wood, biofuel or paper. In addition, the invention provides the use of acell or a plant of the invention in altering the mechanical propertiesof a plant or a plant derived product.

In a further aspect, the present invention provides a method ofmanipulating the growth and/or structure of a plant, comprising thesteps of:

-   -   i) providing a cell/seed according to the invention;    -   ii) regenerating said cell/seed into a plant; and optionally    -   iii) monitoring the levels of CLE41 and/or CLE42 or a receptor        thereof, and or PXY or functional equivalents thereof in said        regenerated plant.

In a further aspect, there is provided an expression constructcomprising a first nucleic acid sequence selected from the groupconsisting of:

-   -   i) a nucleic acid molecule that encodes a CLE41 polypeptide;    -   ii) a nucleic acid molecule that encodes a CLE42 polypeptide;    -   iii) a nucleic acid molecule that encodes a polypeptide        comprising an amino acid sequence that is at least 70% identical        to the amino acid sequence of amino acids 124 to 137 of the        consensus sequence of FIG. 10 (SEQ ID NO. 12);    -   iv) a nucleic acid molecule which is at least 70% identical to        the nucleotide sequence of CLE41 of FIG. 13B (SEQ ID NO. 22) or        CLE42 of FIG. 14B (SEQ ID NO. 24);    -   v) a nucleic acid molecule that hybridizes under stringent        conditions to the nucleotide sequence i) or ii);        and optionally a second nucleic sequence encoding a regulatory        sequence capable of expressing the first nucleic sequence        specifically in or adjacent to the vascular tissue of a plant.

Preferably, the regulatory sequence will be capable of directingexpression of a nucleotide sequence specifically to the vascular tissue,preferably to the cambial/procambial cells and more preferably to tissueadjacent to the cambial/procambial cells i.e. the phloem and/or xylemtissue. Most preferably, a regulatory sequence used in the presentinvention will be capable of directing expression specifically to thephloem cells. Examples of suitable phloem specific regulatory sequencesare SUC2 and APL, KAN1, KAN2, At4g33660, At3g61380, At1g79380. Xylemspecific regulatory sequences may also be used in the present invention.Examples include REV, IRX1 COBL4, KOR, At2g38080, and At1g27440, thepromoter sequence for the irregular xylem3 (irx3) (AtCESA7) gene, thepromoter sequence for the irregular xylem7 (FRAGILE FIBER 8) gene, andthe promoter sequence for the irregular xylem12 (ARABIDOPSISLACCASE-LIKE MULTICOPPER OXIDASE 4) gene (Brown et al. The Plant Cell,Vol. 17, 2281-2295).

Optionally, the expression cassette may further comprise a third nucleicacid sequence selected from the group consisting of:

-   -   i) a nucleic acid molecule that encodes a CLE41 receptor;    -   ii) a nucleic acid molecule that encodes a CLE42 receptor;    -   iii) a nucleic acid molecule that encodes a polypeptide        comprising an amino acid sequence that is at least 70% identical        to the consensus sequence of FIG. 12 (SEQ ID NO. 20), or a        functional equivalent thereof;    -   iv) a nucleic acid molecule that is at least 70% identical to        the nucleotide sequence of FIG. 15B (SEQ ID NO. 26) or 15C (SEQ        ID NO. 27);    -   v) a nucleic acid molecule that is at least 70% identical to a        nucleic acid molecule of i) or ii);    -   vi) a nucleic acid molecule that hybridizes under stringent        conditions to the nucleotide sequence i) or ii)

Preferably, the expression cassette comprises a nucleic acid encodingPXY or a functional equivalent thereof. The third nucleic acid sequencemay be provided on the same expression cassette as the first and/orsecond nucleic acid sequence, or on a separate expression cassette tothe first nucleic acid sequence. The third nucleic acid sequence may beunder the control of fourth nucleic acid sequence encoding a regulatorysequence capable of expressing the third nucleic sequence specificallyin or adjacent to the vascular tissue of a plant.

The second nucleic acid sequence may be the same or different to thefourth nucleic acid sequence.

In a further aspect, there is provided a host cell or organismcomprising an expression construct of the invention.

According to a further aspect of the invention there is provided atransgenic plant seed comprising a cell according to the invention.

The present invention also provides a plant derived product produced bya method of the invention.

The present invention also provides a host cell or organism comprisingan expression construct of the invention. A host cell or organism may bea plant cell, plant seed, plant, or other plant material.

The present a method of producing a plant-derived product comprising:

-   -   a) manipulating the growth and/or structure of a plant using the        methods of the invention;    -   b) growing the plant until it reaches a pre-determined lateral        size; optionally    -   c) harvesting the plant derived product of the plant.

A plant-derived product may include biomass, fibres, forage,biocomposites, biopolymers, wood, biofuel or paper.

The present invention also provides a method of altering the mechanicalproperties of a plant or plant derived product comprising:

-   -   a) manipulating the growth and/or structure of a plant using the        methods of the invention;    -   b) growing the plant until it reaches a pre-determined size; and        optionally    -   c) harvesting a plant derived product of the plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the cambial meristem of birch (A) and Arabidopsis (B). (A)Transverse section of the growing cambium in Birch. (B) Cross section ofa vascular bundle from an Arabidopsis stem. Phloem (ph), xylem (xy),procambium (pc) and cambium (c) are indicated.

FIG. 2 is a diagrammatic representation of vascular development by theprocambium (in, for example, Aribidopsis) or by the cambium (for examplein trees such as birch). New cells arise from division of procambialcells that subsequently differentiate into the phloem of xylem. In thismodel a ligand expressed in the xylem signals cell division in theprocambium.

FIG. 3 shows the effects of over-expressing CLE41 (B,C) and CLE42 (D) onArabidopsis vascular development compared to the wild type (A) in 35 dayold plants. See also Table 1.

FIG. 4 is a graph showing the average number of cells in the vascularbundles of wild type compared to 35S::CLE41 and 35S::CLE42 Arabidopsisplants at 35 days.

FIG. 5 shows the effect of over-expressing CLE41 on the stem vascularbundle of an Arabidopsis compared to the wild-type. Sections throughstem vascular bundles of wild type, 35S::CLE41 and 35S::CLE42 from 50day old plants. A large number of the extra cells in 35S::CLE41 plants(c.f. FIGS. 3 and 4) have differentiated into xylem cells. 35S::CLE41therefore has more xylem cells than wild type.

FIG. 6 shows the effects on plant stature of wild type compared to35S::CLE41 and 35S::CLE42 in Arabidopsis.

FIG. 7 shows the disrupted hypocotyl structure of a Arabidopsis plantwhere either CLE41 (M) or CLE42 (N) is over-expressed and compared withthe wild type (L). In the transgenic lines (M, N), hypocotyls are muchlarger.

FIG. 8 shows the effect of over expressing both CLE41 and/or CLE42 andPXY on the structure and amount of cells in the vascular bundle andinterfascicular region of stems from an Arabidopsis thaliana plant.Simultaneous over-expression of PXY and CLE41/42 gives increased invascular cell number compared to plants over expressing CLE41/42 alone(c.f. FIG. 3).

FIG. 9 shows the effect of over expression of both CLE41 and/or CLE42and PXY on the leaf structure of an Arabidopsis thaliana plant. Multiplemidveins in 35S::CLE42 35S::PXY plants demonstrate that expression ofCLE42 and PXY can initiate vascular tissue.

FIG. 10 is an alignment of rice, poplar and Arabidopsis thalianaputative PXY ligands: 1395 (SEQ ID NO. 1); 5110 (SEQ ID NO. 2); Cle41(SEQ ID No. 3); Cle 44 (SEQ ID NO. 4); 148 (SEQ ID NO. 5);1849 (SEQ IDNO. 6); 428 (SEQ ID NO. 7); Cle46 (SEQ ID NO. 8); OsCLE205 (SEQ ID NO.9); Cle42 (SEQ ID NO. 10); OsCLE102 (SEQ ID NO. 11); and consensus (SEQID NO. 12).

FIG. 11 shows the conservation of residues in the CLE signallingdomain—the dashed line indicates the group 5 that contains all theputative PXY ligands.

FIG. 12 is a comparison of the kinase domain of PXY (Arabidopsisthaliana) from proteins in rice (Os02g02140.1), poplar (PttPXY) andArabidopsis thaliana (PXL1 and PXL2): Os02g02140 (SEQ ID NO. 13);Os03g05140 (SEQ ID NO. 14); OsPXY (SEQ ID NO. 15); PttPXY (SEQ ID NO.16); PXY (SEQ ID NO. 17); PXL1 (SEQ ID NO. 18); PXL2 (SEQ ID NO. 19);and Consensus (SEQ ID NO. 20).

FIG. 13 shows the amino acid sequence of the CLE41 proteins (A) (SEQ IDNO. 21), and nucleotide sequence of the CLE41 gene (B) (SEQ ID NO. 22).

FIG. 14 shows the amino acid sequence of the CLE42 proteins (A) (SEQ IDNO 23) and nucleotide sequence of the CLE42 gene (B) (SEQ ID NO. 24).

FIG. 15 shows the amino acid sequence of the PXY proteins (A) (SEQ IDNO. 25).and nucleotide sequence of the PXY gene without (B) (SEQ ID NO.26) or with (C) the intron (SEQ ID NO. 27).

FIG. 16 shows preferred promoter and terminator sequences for use in theinvention: 1.35S promoter (SEQ ID NO. 28); IRX3 promoter (SEQ ID NO.29); LRR promoter (SEQ ID NO. 30); Nodulin promoter (SEQ ID NO. 31);1.35S terminator (SEQ ID NO. 32) and NOS terminator (SEQ ID NO. 33).

FIG. 17 shows the diagram of the multisite gateway kit for cloning.

FIG. 18 is an alignment of full length PXY and related PXY proteins frompoplar (PttPXY) and Arabidopsis thaliana (PXYL1 and 2): CLV-1 (SEQ IDNO. 34); PXY (SEQ ID NO. 35); PXYL-1 (SEQ ID NO. 36); PXYL-2 (SEQ ID NO.37); OsPXY (SEQ ID NO. 38); PttPXY (SEQ ID NO. 39) and consensus (SEQ IDNO. 40).

FIG. 19 the heights of Nicotiana plants which over express CLE41, PXY orboth are shown. A height defect is associated with plants carrying the35S::CLE41 construct. Normal plant height is restored when plantsharbour both 35S::CLE41 and 35S::PXY cassettes.

FIG. 20 shows the cross section of the Nicotiana plants showing tissuestructure and size. 35S::CLE41 35S::PXY plants have hypocotyls largerthan wild type

FIG. 21 shows cell organisation in Nicotiana plants in transversesection. 35S::CLE41 and 35S::CLE42 plants have more vascular tissue thanwild type, but it is not ordered. 35S::CLE41 35S::PXY plants haveordered vasculature. Given that these plants have larger hypocotyls thanwild type (see FIG. 20), and are of normal height (see FIG. 19), theseplants clearly have more vascular tissue than wild type.

FIG. 22 shows the effect of phloem specific promoter SUC2 and xylemspecific promoter IRX3 on cell organisation in Arabidopsis. IRX3::CLE41plants have large vascular bundles. IRX3::CLE41 35S::PXY plants havelarge vascular bundles with large amounts of secondary growth (c.f. FIG.8 wild type). SUC2::CLE41 and SUC2::CLE41 35S::PXY plants have vasculartissue that is highly ordered with many more vascular cells than wildtype.

FIG. 23 is a graph showing the mean number of cells per vascular bundle.Expression of CLE41 in phloem cells under SUC2 gives more cells pervascular bundle in plants 6 week old Arabidopsis plants.

FIG. 24 is a graph showing the effect of over expression of CLE41 andCLE42 on hypocotyl diameter in Arabidopsis.

FIG. 25 shows the effect of over expression of CLE41 in poplar treesusing the SUC2 or 35S promoters. The bracket denotes xylem cells, ofwhich there are more in 35S::CLE41 and SUC2::CLE41 than wild type.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the finding that CLE41 and CLE42function as ligands for the transmembrane receptor kinase PXY in plants,and modify and/or initiate the regulatory pathway which controls celldivision and differentiation in the vascular tissue of a plant. Thus, bymodulating the levels of CLE41 and/or CLE42 in a plant, optionally incombination with PXY, the growth and/or structure of the plant can bemanipulated, as hereinbefore described.

In particular, the present invention is based upon the finding thatindividual over-expression of CLE41 and/or CLE42 leads to an excess ofundifferentiated cells in the vascular meristem and a subsequentincrease in the radial thickness of the xylem. Further, over-expressionof PXY or a functional equivalent thereof, together with a PXY ligandsuch as CLE41 and/or CLE42, results in an excess of undifferentiatedcells in the vascular meristem of the plant, which show a highly orderedstructure. This excess of cells in the vascular meristem have been shownto then differentiate into xylem cells, thus increasing the radialthickness of the xylem and the biomass of the plant.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of without abolishing or, more preferably,without substantially altering a biological activity, whereas an“essential” amino acid residue results in such a change. For example,amino acid residues that are conserved among the polypeptides of thepresent invention, e.g., those present in the conserved potassiumchannel domain are predicted to be particularly non-amenable toalteration, except that amino acid residues in transmembrane domains cangenerally be replaced by other residues having approximately equivalenthydrophobicity without significantly altering activity.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),non-polar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, anonessential amino acid residue in protein is preferably replaced withanother amino acid residue from the same side chain family.Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of coding sequences, such as by saturationmutagenesis, and the resultant mutants can be screened for biologicalactivity to identify mutants that retain activity. Following mutagenesisof a polypeptide, the encoded proteins can be expressed recombinantlyand the activity of the protein can be determined.

As used herein, a “biologically active fragment” of protein includesfragment of protein that participate in an interaction between moleculesand non-molecules. Biologically active portions of protein includepeptides comprising amino acid sequences sufficiently homologous to orderived from the amino acid sequences of the protein, which includefewer amino acids than the full length protein, and exhibit at least oneactivity of protein. Typically, biologically active portions comprise adomain or motif with at least one activity of the protein, e.g., theability to modulate membrane excitability, intracellular ionconcentration, membrane polarization, and action potential.

As used herein, the term “nucleic acid molecule” includes DNA molecules(e.g., a cDNA or genomic DNA) and RNA molecules (e.g., a mRNA) andanalogs of the DNA or RNA generated, e.g., by the use of nucleotideanalogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA. The nucleotidesequence may be RNA or DNA, including cDNA.

With regards to genomic DNA, the term “isolated” includes nucleic acidmolecules that are separated from the chromosome with which the genomicDNA is naturally associated. Preferably, an “isolated” nucleic acid isfree of sequences that naturally flank the nucleic acid (i.e., sequenceslocated at the 5′- and/or 3′-ends of the nucleic acid) in the genomicDNA of the organism from which the nucleic acid is derived. Moreover, an“isolated” nucleic acid molecule, such as a cDNA molecule, can besubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein).

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which include an open reading frame encoding protein, andcan further include non-coding regulatory sequences and introns.

In the present invention, CLE41 and CLE42 are polypeptides comprisingthe amino acid sequence of FIG. 13A (SEQ ID NO. 21) or 14A (SEQ ID NO.23), respectively. CLE41 and CLE42 are ligands which are able toactivate a kinase receptor, and result in phosphorylation of itself orits target. References to CLE41 and/or CLE42 include functionalequivalents thereof. By a functional equivalent of CLE41 or CLE42 ismeant a polypeptide which is derived from the consensus sequence of FIG.10 by addition, deletion or substitution of one or more amino acids,preferably non-essential amino acids. Preferably, a substitution is aconservative substitution. A functional equivalent of CLE41 and/or CLE42for use in the present invention will be biologically active, andpreferably have some or all of the desired biological activity of thenative polypeptide, preferably the ability to bind to PXY or afunctional equivalent thereof and regulate growth and/or differentiationof the vascular tissue. Preferably, the equivalent is a signallingprotein, preferably of less than 15 kDa in mass, and preferablycomprising a hydrophobic region at the amino terminus. Functionalequivalents may exhibit altered binding characteristics to PXY comparedto a native CLE41 and/or CLE42 protein, but will mediate the samedownstream signalling pathway. Preferred functional equivalents may showreduced non-desirable biological activity compared to the native proteinPreferably, the equivalent comprises a conserved region of 14 aminoacids having a sequence which is at least 70% more preferably 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the conserved regionof amino acids 124 to 137 of the consensus of FIG. 10 (SEQ ID NO. 12),and more preferably across the full length of the consensus sequence. Afunctional equivalent of CLE41 and/or CLE42 preferably also shares atleast 50%, even more preferably at least 60%, and even more preferablyat least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology withCLE41 or CLE42. Preferably, a functional equivalent may also sharesequence identity with the CLE41 and/or CLE42 sequence of FIGS. 13A (SEQID NO. 21) and 14A (SEQ ID NO. 23), respectively. Preferably, functionalequivalents have at least preferably at least 50%, even more preferablyat least 60%, and even more preferably at least 70%, 75%, 80%, 82%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% % sequence identity with the amino acid sequence of FIGS.13A (SEQ ID NO. 21) and 14A (SEQ ID NO. 23) respectively.

References to CLE41 and/or CLE42 also include fragments of the CLE41and/or CLE42 polypeptides or their functional equivalents. A fragment isa portion of a polypeptide sequence, preferably which retains some orall of the biological activity of the full length sequence. Preferably,fragments of CLE41 and/or CLE42 retain the ability to bind PXY andregulate the growth and/or differentiation of the vascular tissue of aplant. Preferably, a fragment may be at least 7 amino acids in length,preferably at least 8, 9, or 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90or 100 amino acids in length, up to the full length CLE polypeptide.Most preferably, a fragment will comprise the conserved regionconsisting of amino acids 124 to 137 of the consensus sequence of FIG.10 (SEQ ID NO. 12).

Nucleic acid molecules encoding CLE41 and CLE42 are preferably thosewhich encode an amino acid sequence as defined by the consensus sequenceof FIG. 10 (SEQ ID NO. 12), and preferably having the sequences as shownin FIG. 13B (SEQ ID NO. 22) or 14B (SEQ ID NO. 24). References tonucleic acid molecules encoding CLE41 and CLE42 also include variants ofthe sequences of FIGS. 13B (SEQ ID NO. 22) or 14B (SEQ ID NO. 24). Avariant sequence is derived from the sequence of FIG. 13B (SEQ ID NO.22) or 14B (SEQ ID NO. 24) by the addition, deletion or substitution ofone or more nucleotide residues. The variant preferably encodes apolypeptide having CLE41 or CLE42 or a functional equivalent thereof, asdefined herein. Preferably, a variant of a nucleotide sequence of FIG.13B (SEQ ID NO. 22) or 14B (SEQ ID NO. 24) will have at least 50%, evenmore preferably at least 60%, and even more preferably at least 70%,75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence ofFIG. 13B (SEQ ID NO. 22) or 14B (SEQ ID NO. 24). Alternatively, avariant sequence which is substantially identical to a sequence of FIG.13B (SEQ ID NO. 22) or 14B (SEQ ID NO. 24) may also be defined as onewhich hybridises under stringent conditions to the complement of anucleotide sequence of FIG. 13B (SEQ ID NO. 22) or 14B (SEQ ID NO. 24).

Nucleic acid molecules encoding CLE41 and/or CLE42 may be derived fromArabidopsis, or may be derived from any other plant and will preferablyshare preferably at least 50%, even more preferably at least 60%, andeven more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity with a CLE41 and/or CLE42 gene from Arabidopsisthaliana, as shown in FIG. 13B (SEQ ID NO. 22) or 14B (SEQ ID NO. 24).

Also encompassed by the present invention are fragments of the nucleicacid molecules encoding CLE41 and/or or CLE42. Preferably, suchfragments encode a fragment of a CLE41 or CLE42 polypeptide as definedherein. A fragment of a nucleic acid molecules encoding CLE41 or CLE42will preferably comprise at least 21 nucleotides in length, morepreferably at least 24, 27, 30 or 33 nucleotides, up to the total numberof nucleotide residues in a full length sequence of FIG. 13B (SEQ ID NO.22) or 14B (SEQ ID NO. 24).

In the present invention, PXY is a polypeptide having the amino acidsequences shown in FIG. 15A (SEQ ID NO. 25). References to PXY includefunctional equivalents thereof. By a functional equivalent of PXY ismeant a polypeptide which is derived from the native PXY polypeptidesequence of FIG. 15 by addition, deletion or substitution of one or moreamino acids. A functional equivalent of PXY for use in the presentinvention will be biologically active, and preferably have some or allof the desired biological activity of the native polypeptide, preferablythe ability to bind to CLE41 and/or CLE42 and regulate growth and/ordifferentiation of the vascular tissue. Functional equivalents mayexhibit altered binding characteristics to CLE41 and/or CLE42 comparedto a native PXY protein. Preferred functional equivalents may showreduced non-desirable biological activity compared to the nativeprotein.

In the present invention, PXY and functional equivalents thereof areproteins found in undifferentiated procambial cells, which mediateactivation of a signalling pathway when bound by CLE41 and/or CLE42,resulting in division of the cambial cells. Preferably, PXY or itsfunctional equivalents is a protein kinase, preferably comprising aleucine rich domain. More preferably, it comprises a LLR-RLK (LeucineRich Repeat-Receptor-Like-Kinase) protein. Preferably, PXY or itsfunctional equivalents are members of the XI family of Arabidopsisthaliana RLK proteins, and preferably comprise a conserved region in thekinase domain having the sequence comprising the consensus sequence ofFIG. 12 (SEQ ID NO. 20) or a biologically active portion thereof, or asequence having at least 30%, 40%, 50%, 55%, 60, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to theconsensus sequence of FIG. 12 (SEQ ID NO. 20). Most preferably, afunctional equivalent thereof will preferably comprise an amino acidhaving at least 70% sequence identity to the consensus sequence of FIG.12 (SEQ ID NO. 20) and preferably will bind a CLE 41 and/or CLE 42polypeptide or fragment thereof. A functional equivalent of PXYpreferably also shares preferably at least 50%, even more preferably atleast 60%, and even more preferably at least 70%, 75%, 80%, 82%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence homology with native PXY. Preferably, a functionalequivalent may also share sequence identity with the PXY sequence ofFIG. 15A (SEQ ID NO. 25), respectively

References to PXY also include fragments of the PXY polypeptides or itsfunctional equivalents. A fragment is a portion of a polypeptidesequence, preferably which retains some or all of the biologicalactivity of the full length sequence. Preferably, fragments of PXYretain the ability to bind a ligand and regulate the growth and/ordifferentiation of the vascular tissue of a plant. Preferably, afragment will comprise at least a portion of the kinase domain,preferably a biologically active portion thereof, up to the full lengthkinase domain. Most preferably, a fragment will further comprise atleast a portion of the extracellular domain, and will preferablycomprise at least a portion of the LLR region.

Nucleic acid molecules encoding PXY are preferably those which encode anamino acid sequence as defined in FIG. 15A (SEQ ID NO. 25), andpreferably having the sequences as shown in FIG. 15B (SEQ ID NO. 26) orC (SEQ ID NO. 27). References to nucleic acid molecules encoding PXYalso include variants of the sequences of FIG. 15B (SEQ ID NO. 26) or C(SEQ ID NO. 27). A variant sequence is a nucleic acid molecules which isderived from the sequence of FIG. 15B (SEQ ID NO. 26) or C (SEQ ID NO.27) by the addition, deletion or substitution of one or more nucleotideresidues. The variant preferably encodes a polypeptide having PXYactivity, as defined herein. Preferably, a variant of a nucleotidesequence of FIG. 15B (SEQ ID NO. 26) or C (SEQ ID NO. 27) will have atleast 50%, even more preferably at least 60%, and even more preferablyat least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with asequence of FIG. 15B (SEQ ID NO. 26) or C (SEQ ID NO. 27).Alternatively, a variant sequence which is substantially identical to asequence of FIG. 15B (SEQ ID NO. 26) or C (SEQ ID NO. 27) may also bedefined as one which hybridises under stringent conditions to thecomplement of a nucleotide sequence of FIG. 15B (SEQ ID NO. 26) or C(SEQ ID NO. 27).

Nucleotide sequences encoding PXY may be derived from Arabidopsis, ormay be derived from any other plant and will preferably share preferablyat least 50%, even more preferably at least 60%, and even morepreferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% % sequenceidentity with a PXY gene from Arabidopsis thaliana, as shown in FIG. 15B(SEQ ID NO. 26) or C (SEQ ID NO. 27). Genbank references arePXY=At5g61480 (TAIR), PXL1=At1g08590 (TAIR), PXL2=At4g28650 (TAIR).

Also encompassed by the present invention are fragments of the nucleicacid molecule encoding PXY. Preferably, such fragments encode a fragmentof a PXY polypeptide as defined herein. A fragment of a nucleic acidmolecule encoding PXY will preferably comprise at least 10, 20, 30, 40,50, 60, 70, 80 or 90 or 100, 200 or 300 or more nucleotides in length,up to the total number of nucleotide residues in a full length sequenceof FIG. 15A (SEQ ID NO. 25).

Also provided in the present invention are antisense sequences of theabove mentioned nucleic acid molecules, which hybridise under stringentconditions to the nucleotide sequences encoding CLE41 and/or CLE42 orPXY, or a functional equivalents thereof, as defined above. Suchsequences are useful in down regulating expression of the CLE41 and/orCLE42 and/or PXY or functional equivalents thereof. Whilst in apreferred embodiment, both receptor and ligand will be eitherup-regulated (over-expressed) or down-regulated in a cell of a plant, itis envisaged that it may in certain circumstances be desirable toup-regulate either the receptor whilst down-regulating the ligand, orvice versa.

As used herein, the term “hybridizes under stringent conditions”describes conditions for hybridization and washing. Stringent conditionsare known to those skilled in the art and can be found in availablereferences (e.g., Current Protocols in Molecular Biology, John Wiley &Sons, N.Y., 1989, 6.3.1-6.3.6). Aqueous and non-aqueous methods aredescribed in that reference and either can be used. A preferred exampleof stringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% (w/v) SDS at 50° C. Another example of stringenthybridization conditions are hybridization in 6×SSC at about 45° C.,followed by one or more washes in 0.2×SSC, 0.1% (w/v) SDS at 55° C. Afurther example of stringent hybridization conditions are hybridizationin 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC,0.1% (w/v) SDS at 60° C. Preferably, stringent hybridization conditionsare hybridization in 6×SSC at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% (w/v) SDS at 65° C. Particularly preferredstringency conditions (and the conditions that should be used if thepractitioner is uncertain about what conditions should be applied todetermine if a molecule is within a hybridization limitation of theinvention) are 0.5 molar sodium phosphate, 7% (w/v) SDS at 65° C.,followed by one or more washes at 0.2×SSC, 1% (w/v) SDS at 65° C.Preferably, an isolated nucleic acid molecule of the invention thathybridizes under stringent conditions to the sequence of FIG. 15 B (SEQID NO. 26) or C (SEQ ID NO. 27) corresponds to a naturally-occurringnucleic acid molecule.

Sequence identity is determined by comparing the two aligned sequencesover a pre-determined comparison window, and determining the number ofpositions at which—identical residues occur. Typically, this isexpressed as a percentage. The measurement of sequence identity of anucleotide sequences is a method well known to those skilled in the art,using computer implemented mathematical algorithms such as ALIGN(Version 2.0), GAP, BESTFIT, BLAST (Altschul et al J. Mol. Biol. 215:403 (1990)), FASTA and TFASTA (Wisconsin Genetic Software PackageVersion 8, available from Genetics Computer Group, Accelrys Inc. SanDiego, Calif.), and CLUSTAL (Higgins et al., Gene 73: 237-244 (1998)),using default parameters.

Calculations of sequence homology or identity between sequences areperformed as follows:

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of thelength of the reference sequence. The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman et al., (1970) J.Mol. Biol. 48:444-453) algorithm which has been incorporated into theGAP program in the GCG software package (available athttp://www.gcg.com), using either a BLOSUM 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6. In yet another preferred embodiment, the percentidentity between two nucleotide sequences is determined using the GAPprogram in the GCG software package (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred setof parameters (and the one that should be used if the practitioner isuncertain about what parameters should be applied to determine if amolecule is within a sequence identity or homology limitation of theinvention) are a BLOSUM 62 scoring matrix with a gap penalty of 12, agap extend penalty of 4, and a frameshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of Meyers et al., (1989) CABIOS4:11-17) which has been incorporated into the ALIGN program (version2.0), using a PAM120 weight residue table, a gap length penalty of 12and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al., (1990) J. Mol. Biol. 215:403-410). BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to nucleic acidmolecules of the invention. BLAST protein searches can be performed withthe XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to protein molecules of the invention. To obtaingapped alignments for comparison purposes, gapped BLAST can be utilizedas described in Altschul et al. (1997, Nucl. Acids Res. 25:3389-3402).When using BLAST and gapped BLAST programs, the default parameters ofthe respective programs (e.g., XBLAST and NBLAST) can be used. See<http://www.ncbi.nlm.nih.gov>.

Sequence comparisons are preferably made over the full-length of therelevant sequence described herein.

The polypeptide sequences and nucleic acid molecules used in the presentinvention may be isolated or purified. By “purified” is meant that theyare substantially free from other cellular components or material, orculture medium. “Isolated” means that they may also be free of naturallyoccurring sequences which flank the native sequence, for example in thecase of nucleic acid molecule, isolated may mean that it is free of 5′and 3′ regulatory sequences.

The polypeptide and nucleic acid molecule used in the invention may benaturally occurring or may be synthetic. The nucleic acid molecule maybe recombinant.

The present invention is based upon using either CLE41 and/or CLE42and/or a PXY, or functional equivalents thereof, to manipulate thegrowth and/or structure of a plant. By “manipulate” is meant alteringthe native growth pattern of a plant, compared to that of anon-manipulated plant of the same species, grown under identicalconditions. The manipulation is preferably effected by altering thelevels of said receptor and ligand in a cell of the plant.

A plant having increased levels of said CLE41 and/or CLE42 and/or a PXY,or functional equivalents thereof in a particular tissue and at apre-selected developmental stage, compared to the native levels in thesame tissue of a native plant of the same species, at the samedevelopmental stage and grown in identical conditions.

Herein, the growth of a plant refers to the size of a plant, preferablythe secondary growth, and preferably the amount of vascular and/orinterfasicular tissue, more preferably the amount of xylem cells, alsoreferred to as the woody tissue or biomass of a plant.

By identical conditions is meant conditions which are the substantiallythe same in terms of temperature, light, and availability of nutrientsand water. By substantially is meant that the conditions may varyslightly, but not to an extent to which is known to affect the growth ofa plant.

The structure of a plant refers to the organisation of tissue in aplant, preferably the vascular tissue, most preferably the polarity ofthe phloem and xylem cells.

The use of said CLE41 and/or CLE42 and/or a PXY, or functionalequivalents thereof as defined herein to manipulate the growth and/orstructure of a plant may be achieved in any manner which alters theregulation of the signalling pathway mediated by CLE41 and/or CLE42binding to PXY. Preferably, the invention may be achieved in any mannerwhich up-regulates the signalling pathway. Preferably, the manipulationis mediated via a PXY ligand as defined herein, preferably CLE41 and/orCLE42, or via a CLE41 and/or CLE42 receptor, preferably PXY. Forexample, manipulation may comprise altering their expression patternwithin the plant, altering the amount of said receptor and/or ligandwithin the plant, or altering the binding pattern thereof.

By modulation of the levels of the CLE41 and/or CLE42 and/or a PXY, orfunctional equivalents thereof, is meant an increase or decrease in thelevels of in the plant, preferably the levels localised in the vasculartissue, and preferably in the cambium or procambium of a plant, ascompared to the levels in the same tissue in a native plant of the samespecies at the same stage if developed and grown under identicalconditions, and in which no modulation has been made. Preferably, thelevels of CLE41 and/or CLE42 and/or a PXY, or functional equivalentsthereof, are increased. Preferred levels of PXY ligand are at least 5,10, 20, 30, 40, 50, 60, 70, 80, 90% more or less relative to said nativeplant. Preferred levels of CLE41 and/or CLE42 receptor are 5, 10, 20,30, 40, 50, 60, 70, 80, 90% more or less relative to said native plant.

The alteration in levels of CLE41 and/or CLE42 and/or a PXY, orfunctional equivalents thereof, as defined above preferably increases ordecreases the activity by at least about 2-fold compared to a basallevel of activity. More preferably said activity is increased ordecreased by at least about 5 fold; 10 fold; 20 fold, 30 fold, 40 fold,50 fold. Preferably said activity is increased or decreased by betweenat least 50 fold and 100 fold. Preferably said increase or decrease isgreater than 100-fold.

It will be apparent that means to modulate the activity of a polypeptideencoded by a nucleic acid molecule are known to the skilled artisan. Forexample, and not by limitation, altering the gene dosage by providing acell with multiple copies of said gene or its complement. Alternatively,or in addition, a gene(s) may be placed under the control of a powerfulpromoter sequence or an inducible promoter sequence to elevateexpression of mRNA encoded by said gene. The modulation of mRNAstability is also a mechanism used to alter the steady state levels ofan mRNA molecule, typically via alteration to the 5′ or 3′ untranslatedregions of the mRNA.

It is envisaged that where a plant naturally expresses said CLE41 and/orCLE42 receptor and/or PXY ligand, their modulation may be achieved byaltering the expression pattern of the native gene(s) and/or productionof the polypeptide. This may be achieved by any suitable method,including altering transcription of the gene, and/or translation of themRNA into polypeptide, and post-translational modification of thepolypeptide.

Altering the expression pattern of a native gene may be achieved byplacing it under control of a heterologous regulatory sequence, which iscapable of directing the desired expression pattern of the gene.Suitable regulatory sequences are described herein. Alternatively,regulation of expression of the native gene may be altered throughchanging the pattern of transcription factors which mediate expressionof the gene. This may require the use of modified transcription factors,whose binding pattern is altered to obtain a desired expression patternof the gene. Alternatively, the copy number of the native gene may beincreased or decreased, in order to change the amount of expression ofthe gene. Suitable methods for carrying out these embodiments of theinvention are known to persons skilled in the art, and may employ theuse of an expression construct according to the invention.

Plants transformed with a nucleic acid molecule or expression constructof the invention may be produced by standard techniques known in the artfor the genetic manipulation of plants. DNA can be introduced into plantcells using any suitable technology, such as a disarmed Ti-plasmidvector carried by Agrobacterium exploiting its natural genetransferability (EP-A-270355, EP-A-0116718, NAR 12(22):8711-87215(1984), Townsend et al., U.S. Pat. No. 5,563,055); particle ormicroprojectile bombardment (U.S. Pat. No. 5,100,792, EP-A-444882,EP-A-434616; Sanford et al, U.S. Pat. No. 4,945,050; Tomes et al. (1995)“Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment”, in Plant Cell, Tissue and Organ Culture: FundamentalMethods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabeet al. (1988) Biotechnology 6: 923-926); microinjection (WO 92/09696, WO94/00583, EP 331083, EP 175966, Green et al. 91987) Plant Tissue andCell Culture, Academic Press, Crossway et al. (1986) Biotechniques4:320-334); electroporation (EP 290395, WO 8706614, Riggs et al. (1986)Proc. Natl. Acad. Sci. USA 83:5602-5606; D'Halluin et al. 91992). PlantCell 4:1495-1505) other forms of direct DNA uptake (DE 4005152, WO9012096, U.S. Pat. No. 4,684,611, Paszkowski et al. (1984) EMBO J.3:2717-2722); liposome-mediated DNA uptake (e.g. Freeman et al (1984)Plant Cell Physiol, 29:1353); or the vortexing method (e.g. Kindle(1990) Proc. Nat. Acad. Sci. USA 87:1228). Physical methods for thetransformation of plant cells are reviewed in Oard (1991) Biotech. Adv.9:1-11. See generally, Weissinger et al. (1988) Ann. Rev. Genet.22:421-477; Sanford et al. (1987) Particulate Sciences and Technology5:27-37; Christou et al. (1988) Plant Physiol. 87:671-674; McCabe et al.(1988) Bio/Technology 6:923-926; Finer and McMullen (1991) In Vitro CellDev. Biol. 27P:175-182; Singh et al. (1988) Theor. Appl. Genet.96:319-324; Datta et al. (1990) Biotechnology 8:736-740; Klein et al.(1988) Proc. Natl. Acad. Sci. USA 85: 4305-4309; Klein et al. (1988)Biotechnology 6:559-563; Tomes, U.S. Pat. No. 5,240,855; Buising et al.U.S. Pat. No. 5,322,783 and U.S. Pat. No. 5,324,646; Klein et al. (1988)Plant Physiol 91: 440-444; Fromm et al (1990) Biotechnology 8:833-839;Hooykaas-Von Slogteren et al. 91984). Nature (London) 311:763-764;Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349; De Wetet al. (1985) in The Experimental Manipulation of Ovule Tissues ed.Chapman et al. (Longman, New York), pp. 197-209; Kaeppler et al. (1990)Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl.Genet. 84:560-566; Li et al. (1993) Plant Cell Reports 12: 250-255 andChristou and Ford (1995) Annals of Botany 75: 407-413; Osjoda et al.(1996) Nature Biotechnology 14:745-750, all of which are hereinincorporated by reference.

Agrobacterium transformation is widely used by those skilled in the artto transform dicotyledonous species. Recently, there has beensubstantial progress towards the routine production of stable, fertiletransgenic plants in almost all economically relevant monocot plants(Toriyama et al. (1988) Bio/Technology 6: 1072-1074; Zhang et al. (1988)Plant Cell rep. 7379-384; Zhang et al. (1988) Theor. Appl. Genet.76:835-840; Shimamoto et al. (1989) Nature 338:274-276; Datta et al.(1990) Bio/Technology 8: 736-740; Christou et al. (1991) Bio/Technology9:957-962; Peng et al (1991) International Rice Research Institute,Manila, Philippines, pp. 563-574; Cao et al. (1992) Plant Cell Rep. 11:585-591; Li et al. (1993) Plant Cell Rep. 12: 250-255; Rathore et al.(1993) Plant Mol. Biol. 21:871-884; Fromm et al (1990) Bio/Technology8:833-839; Gordon Kamm et al. (1990) Plant Cell 2:603-618; D'Halluin etal. (1992) Plant Cell 4:1495-1505; Walters et al. (1992) Plant Mol.Biol. 18:189-200; Koziel et al. (1993). Biotechnology 11194-200; Vasil,I. K. (1994) Plant Mol. Biol. 25:925-937; Weeks et al (1993) PlantPhysiol. 102:1077-1084; Somers et al. (1992) Bio/Technology10:1589-1594; WO 92/14828. In particular, Agrobacterium mediatedtransformation is now emerging also as an highly efficienttransformation method in monocots. (Hiei, et al. (1994) The PlantJournal 6:271-282). See also, Shimamoto, K. (1994) Current Opinion inBiotechnology 5:158-162; Vasil, et al. (1992) Bio/Technology 10:667-674;Vain, et al. (1995) Biotechnology Advances 13(4):653-671; Vasil, et al.(1996) Nature Biotechnology 14: 702).

Microprojectile bombardment, electroporation and direct DNA uptake arepreferred where Agrobacterium is inefficient or ineffective.Alternatively, a combination of different techniques may be employed toenhance the efficiency of the transformation process, e.g. bombardmentwith Agrobacterium-coated microparticles (EP-A-486234) ormicroprojectile bombardment to induce wounding followed byco-cultivation with Agrobacterium (EP-A-486233).

Altering the production of a polypeptide may be achieved by increasingthe amount of mRNA produced, increasing the stability of protein,altering the rate of post translational modification for examplealtering rates of proteolytic cleavage.

Altering the post-translational modification of a polypeptide may alsoaffect its structure and function, and may be used to alter theexpression of the native polypeptide. For example, the ligand is likelyto be only a portion of the full length proteins and the active ligandis probably released by proteolysis.

Alternatively, a polypeptide or nucleic acid molecule as defined hereinmay be introduced into the plant, by any suitable means such asspraying, uptake by the roots, or injection into phloem. Todown-regulate said receptor or ligand in a plant, an enzyme may beintroduced which inhibits or digests one or both of the receptor orligand.

In addition, modulating the activity mediated by CLE41 and/or CLE42and/or a PXY, or functional equivalents thereof, by altering theirbinding pattern, in order to up-or-down-regulate the downstreamsignalling pathway. The binding pattern may be altered in any suitableway, for example by altering the structure, binding affinity, temporalbinding pattern, selectivity and amount available for binding on thecell surface of CLE41 and/or CLE42 and/or a PXY, or functionalequivalents thereof.

The binding pattern may be altered by making appropriate variations tothe ligand polypeptide, for example to change its binding site to thereceptor, using known methods for mutagenesis.

Alternatively, non-protein analogues may be used. Methods formanipulating a polypeptide used in the present invention are known inthe art, and include for example altering the nucleic acid sequenceencoding the polypeptide. Methods for mutagenesis are well known.Preferably, where variants are produced using mutagenesis of the nucleicacid coding sequence, this is done in a manner which does not affect thereading frame of the sequence and which does not affect the polypeptidein a manner which affects the desired biological activity.

In selecting suitable variants for use in the present invention, routineassays may be used to screen for those which have the desiredproperties. This may be done by visual observation of plants and plantmaterial, or measuring the biomass of the plant or plant material.

Thus, for use in altering the expression of the CLE41 and/or CLE42and/or a PXY, or functional equivalents thereof, in a cell of a plant,there is provided an expression cassette comprising a regulatorysequence to modulate the expression of the native CLE41 and/or CLE42 orPXY genes in a plant. Preferably, the regulatory sequences are designedto be operably linked to the native gene, in order to direct expressionin a manner according to the present invention.

The nucleic acid molecules as described herein, and/or a regulatorysequence are preferably provided as part of an expression cassette, forexpression of the sequence in a cell of interest. Suitable expressioncassettes may also comprise 5′ and 3′ regulatory sequences operablylinked to the sequences of interest. In addition, genes encoding, forexample, selectable markers and reporter genes may be included. Theexpression cassette will preferably also contain one or more restrictionsites, to enable insertion of the nucleotide sequence and/or aregulatory sequence into the plant genome, at a pre-selected position.Also provided on the expression cassette may be transcription andtranslation initiation regions, to enable expression of the incominggenes, transcription and translational termination regions, andregulatory sequences. These sequences may be native to the plant beingtransformed, or may be heterologous and/or foreign.

Heterologous sequences are sequences which in nature are not operablylinked to each other and/or are not found next to each other in a nativesequence. In contrast, homologous sequences refer to sequences whichshare sequence similarity, which may be described as sequence homology.Homology is usually in a fragment of the sequence, typically in afunctional domain of the sequence.

A foreign sequence is one which is not found in the native genome of theplant being transformed.

A regulatory sequence is a nucleotide sequence which is capable ofinfluencing transcription or translation of a gene or gene product, forexample in terms of initiation, rate, stability, downstream processing,and mobility. Examples of regulatory sequences include promoters, 5′ and3′ UTR's, enhancers, transcription factor or protein binding sequences,start sites and termination sequences, ribozyme binding sites,recombination sites, polyadenylation sequences, sense or antisensesequences. They may be DNA, RNA or protein. The regulatory sequences maybe plant- or virus derived, and preferably may be derived from the samespecies of plant as the plant being modulated.

By “promoter” is meant a nucleotide sequence upstream from thetranscriptional initiation site and which contains all the regulatoryregions required for transcription. Suitable promoters includeconstitutive, tissue-specific, inducible, developmental or otherpromoters for expression in plant cells comprised in plants depending ondesign. Such promoters include viral, fungal, bacterial, animal andplant-derived promoters capable of functioning in plant cells.

Constitutive promoters include, for example CaMV 35S promoter (Odell etal. (1985) Nature 313, 9810-812); rice actin (McElroy et al. (1990)Plant Cell 2: 163-171); ubiquitin (Christian et al. (1989) Plant Mol.Biol. 18 (675-689); pEMU (Last et al. (1991) Theor Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3. 2723-2730); ALS promoter(U.S. application Ser. No. 08/409,297), and the like. Other constitutivepromoters include those in U.S. Pat. Nos. 5,608,149; 5,608,144;5,604,121; 5,569,597; 5,466,785; 5,399,680, 5,268,463; and U.S. Pat. No.5,608,142.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducedgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 andMcNellis et al. (1998) Plant J. 14(2): 247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227: 229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156, herein incorporated by reference.

Where enhanced expression in particular tissues is desired,tissue-specific promoters can be utilised. Tissue-specific promotersinclude those described by Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7): 792-803;Hansen et al. (1997) Mol. Gen. Genet. 254(3): 337-343; Russell et al.(1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) PlantPhysiol. 112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol.112(2): 525-535; Canevascni et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5): 773-778; Lam(1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al. (1993)Plant Mol. Biol. 23(6): 1129-1138; Mutsuoka et al. (1993) Proc. Natl.Acad. Sci. USA 90 (20): 9586-9590; and Guevara-Garcia et al (1993) PlantJ. 4(3): 495-50.

“Operably linked” means joined as part of the same nucleic acidmolecule, suitably positioned and oriented for transcription to beinitiated from the promoter. DNA operably linked to a promoter is “undertranscriptional initiation regulation” of the promoter. In a preferredaspect, the promoter is an inducible promoter or a developmentallyregulated promoter.

The promoters which control the expression of CLE41 and/or CLE42 arepreferably tissue or organ specific, such that expression of CLE41and/or CLE42 can be directed to a particular organ or tissue, such asthe vascular tissue, preferably the cambium or procambium, and mostpreferably phloem or xylem tissue. The promoters may be constitutive,whereby they direct expression under most environmental or developmentalconditions. More preferably, the promoter is inducible, and will directexpression in response to environmental or developmental cues, such astemperature, chemicals, drought, and others. The promoter may also bedevelopmental stage specific.

Examples of suitable promoter sequences include those of the T-DNA of A.tumefaciens, including mannopine synthase, nopaline synthase, andoctopine synthase; alcohol dehydrogenase promoter from corn; lightinducible promoters such as ribulose-biphosphate-carboxylase smallsubunit gene from various species and the major chlorophyll a/b bindingprotein gene promoter; histone promoters (EP 507 698), actin promoters;maize ubiquitin 1 promoter (Christensen et al. (1996) Transgenic Res.5:213); 35S and 19S promoters of cauliflower mosaic virus;developmentally regulated promoters such as the waxy, zein, or bronzepromoters from maize; as well as synthetic or other natural promotersincluding those promoters exhibiting organ specific expression orexpression at specific development stage(s) of the plant, like thealpha-tubulin promoter disclosed in U.S. Pat. No. 5,635,618. Preferredphloem specific promoters include SUC2, APL, KAN1, KAN2, At4g33660,At3g61380, and At1g79380. Preferred xylem specific promoters includeREV, IRX1 COBL4, KOR, At2g38080, and At1g2744.

Suitable expression cassettes for use in the present invention can beconstructed, containing appropriate regulatory sequences, includingpromoter sequences, terminator fragments, polyadenylation sequences,enhancer sequences, marker genes and other sequences as appropriate. Forfurther details see, for example, Molecular Cloning: Laboratory Manual:2^(nd) edition, Sambrook et al. 1989, Cold Spring Harbor LaboratoryPress or Current Protocols in Molecular Biology, Second Edition, Ausubelet al. Eds., John Wiley & Sons, 1992. The expression cassettes may be abi-functional expression cassette which functions in multiple hosts. Inthe case of GTase genomic DNA this may contain its own promoter or otherregulatory elements and in the case of cDNA this may be under thecontrol of an appropriate promoter or other regulatory elements forexpression in the host cell.

An expression cassette including a nucleic acid molecule according tothe invention need not include a promoter or other regulatory sequence,particularly if the vector is to be used to introduce the nucleic acidinto cells for recombination into the gene.

Suitable selectable marker or reporter genes may be used to facilitateidentification and selection of transformed cells. These will confer aselective phenotype on the plant or plant cell to enable selection ofthose cells which comprise the expression cassette. Preferred genesinclude the chloramphenicol acetyl transferase (cat) gene from Tn9 of E.coli, the beta-gluronidase (gus) gene of the uidA locus of E. coli, thegreen fluorescence protein (GFP) gene from Aequoria victoria, and theluciferase (luc) gene from the firefly Photinus pyralis. If desired,selectable genetic markers may be included in the construct, such asthose that confer selectable phenotypes such as resistance to antibodiesor herbicides (e.g. kanamycin, hygromycin, phosphinotricin,chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinonesand glyphosate).

Reporter genes which encode easily assayable marker proteins are wellknown in the art. In general, a reporter gene is a gene which is notpresent or expressed by the recipient organism or tissue and whichencodes a protein whose expression is manifested by some easilydetectable property, e.g. phenotypic change or enzymatic activity.

The selectable marker or reporter gene may be carried on a separateexpression cassette and co-transformed with the expression cassette ofthe invention. The selectable markers and/or reporter genes may beflanked with appropriate regulatory sequences to enable their expressionin plants.

The expression cassette may also comprise elements such as introns,enhancers, and polyadenylation sequences. These elements must becompatible with the remainder of the expression cassette. These elementsmay not be necessary for the expression or function of the gene but mayserve to improve expression or functioning of the gene by affectingtranscription, stability of the mRNA, or the like. Therefore, suchelements may be included in the expression construct to obtain theoptimal expression and function of CLE41 and/or CLE42 and/or PXY in theplant.

The expression cassette comprising the heterologous nucleic acid mayalso comprise sequences coding for a transit peptide, to drive theprotein coded by the heterologous gene into a desired part of the cell,for example the chloroplasts. Such transit peptides are well known tothose of ordinary skill in the art, and may include single transitpeptides, as well as multiple transit peptides obtained by thecombination of sequences coding for at least two transit peptides. Onepreferred transit peptide is the Optimized Transit Peptide disclosed inU.S. Pat. No. 5,635,618, comprising in the direction of transcription afirst DNA sequence encoding a first chloroplast transit peptide, asecond DNA sequence encoding an N-terminal domain of a mature proteinnaturally driven into the chloroplasts, and a third DNA sequenceencoding a second chloroplast transit peptide.

In the present invention, any plant species may be used, including bothmonocots and dicots. Preferred plants for use in the present inventionare those which are targets for biomass, and/or are readily grown,exhibit high growth rates, are easily harvested, and can be readilyconverted to a biofuel. Preferred plants include grasses, trees, crops,and shrubs.

Suitable plants for use in the present invention are those which intheir native form produce a high yield of feedstock, for paper or fuelproduction. Examples of suitable plant types include perennial fastgrowing herbaceous and woody plants, for example trees, shrubs andgrasses. Preferred trees for use in the invention include poplar, hybridpoplar, willow, silver maple, black locust, sycamore, sweetgum andeucalyptus. Preferred shrubs include tobacco. Perennial grasses includeswitchgrass, reed canary grass, prairie cordgrass, tropical grasses,Brachypodiumdistachyon, and Miscanthes. Crops include wheat, soybean,alphalpha, corn, rice, maize, and sugar beet.

In yet still a further preferred embodiment of the invention said plantis a woody plant selected from: poplar; eucalyptus; Douglas fir; pine;walnut; ash; birch; oak; teak; spruce. Preferably said woody plant is aplant used typically in the paper industry, for example poplar.

Methods to transform woody species of plant are well known in the art.For example the transformation of poplar is disclosed in U.S. Pat. No.4,795,855 and WO9118094. The transformation of eucalyptus is disclosedin EP1050209 and WO9725434. Each of these patents is incorporated intheir entirety by reference.

In a still further preferred embodiment of the invention said plant isselected from: corn (Zea mays), canola (Brassica napus, Brassica rapassp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secalecerale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower(helianthus annuas), wheat (Tritium aestivum), soybean (Glycine max),tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (lopmoeabatatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut(Cocos nucifera), pineapple (Anana comosus), citrus tree (Citrus spp.)cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musa spp.),avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava),mango (Mangifer indica), olive (Olea europaea), papaya (Carica papaya),cashew (Anacardium occidentale), macadamia (Macadamia intergrifolia),almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley,vegetables and ornamentals.

Preferably, plants of the present invention are crop plants (forexample, cereals and pulses, maize, wheat, potatoes, tapioca, rice,sorghum, millet, cassava, barley, pea, and other root, tuber or seedcrops. Important seed crops are oil-seed rape, sugar beet, maize,sunflower, soybean, and sorghum. Horticultural plants to which thepresent invention may be applied may include lettuce, endive, andvegetable brassicas including cabbage, broccoli, and cauliflower, andcarnations and geraniums. The present invention may be applied intobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper,chrysanthemum.

Grain plants that provide seeds of interest include oil-seed plants andleguminous plants. Seeds of interest include grain seeds, such as corn,wheat, barley, rice, sorghum, rye, etc. Oil-seed plants include cotton,soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut,etc. Leguminous plants include beans and peas. Beans include guar,locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, limabean, fava been, lentils, chickpea, etc.

In a preferred embodiment of the invention said seed is produced from aplant selected from the group consisting of: corn (Zea mays), canola(Brassica napus, Brassica rapa ssp.), flax (Linum usitatissimum),alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cerale),sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthusannus), wheat (Tritium aestivum), soybean (Glycine max), tobacco(Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachishypogaea), cotton (Gossypium hirsutum), sweet potato (lopmoea batatus),cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocosnucifera), pineapple (Anana comosus), citris tree (Citrus spp.) cocoa(Theobroma cacao), tea (Camellia senensis), banana (Musa spp.), avacado(Persea americana), fig (Ficus casica), guava (Psidium guajava), mango(Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew(Anacardium occidentale), macadamia (Macadamia intergrifolia), almond(Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley,vegetables.

The present invention has uses in methods which require increasedbiomass in plants, for example where plant biomass is used in themanufacture of products such as biofuels and paper. The invention is notlimited to methods of making these particular products, and it isenvisaged that the invention will be applicable to the manufacture of avariety of plant based products. In addition, the invention is alsouseful in altering the characteristics of plant material, such that theplant material can be adapted for particular purposes. In one suchembodiment, over expression of the ligand and/or receptor as definedherein may be used to increase the number of cells in the vasculartissue of a plant, but without increasing the actual biomass of theplant (i.e. the number of cells may be increased, but the size of thesecells is smaller). This has the effect of increasing the density of thevascular tissue, and therefore producing a harder wood. Thus, theinvention includes methods for the production of a wood product having aparticular density. In addition, it is envisaged that by manipulatingplant cells to differentiate their vascular tissue, and therefore grow,environmental growth signals may be bypassed and the present inventionmay be used to extend the growth season of plants, beyond that whichwould be possible in a native plant.

The embodiments described in relation to the each aspect apply to theother aspects of the invention, mutatis mutandis.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of the words, for example“comprising” and “comprises”, means “including but not limited to”, andis not intended to (and does not) exclude other moieties, additives,components, integers or steps.

Throughout the description and claims of this specification, thesingular encompasses the plural unless the context otherwise requires.In particular, where the indefinite article is used, the specificationis to be understood as contemplating plurality as well as singularity,unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties orgroups described in conjunction with a particular aspect, embodiment orexample of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith.

The present invention will now be described with reference to thefollowing non-limiting examples:

EXAMPLES

DNA manipulation was carried out using standard methods. Over expression(35S) constructs for plant transformation were generated by cloningCLE41, CLE42, and PXY genomic DNA sequences into pK2GW7,0 (M. Karimi, D.Inze, A. Depicker, Trends in Plant Science 7, 193 (2002)) using gatewaytechnology (invitrogen) with primers listed in the table. Sequences wereamplified by PCR and cloned into pENTR-D-TOPO. Subsequently, reactionscontaining LR clonase II and pK2GW7,0 and the relevant TOPO vector,sequences were used to transfer sequences in pENTR-D-TOPO to the binaryplasmid. pIRX3::CLE41/42 were constructed using the p3HSC Gatewaydestination vector (Atanassov et al. 2008) derived from pCB1300 byinsertion of the 1.7 kb promoter sequences of irx3, the frame A(attR1/cmR/ccdB/attR2) cassette (Invitrogen) and the NOS terminatorregion from pGPTV-BAR. For SUC2::CLE41 we used overlapping PCR. The SUC2promoter and CLE41 coding sequence were amplified separately withoverlapping ends. These products were mixed, annealed and elongatedprior to amplification with SUC2 and CLE41 forward and reverse oligosrespectively. The resulting PCR product was cloned into pTF101.gw1 (Pazet al. 2004) via pENTR-D-TOPO. For tissue specific expression, promotersknown to give xylem (IRX3; (Gardiner et al. 2003) or phloem (SUC2;(Truernit and Sauer 1995) specific expression were used. Plasmids weresequenced and transformed into Arabidopsis using the method of Cloughand Bent (S. J. Clough, A. F. Bent, Plant Journal 16, 735 (December,1998)),

In order to understand if over expression of PXY and CLE41 had the sameeffect in poplar, plants were transformed in tissue culture usingAgrobacterium to transfer the constructs into poplar tissue using themethod of Meilan and Ma (R. Meilan, C. Ma, Methods in Molecular Biology344, 143 (2006). 35S::CLE41, SUC::CLE41 and wild type were grown inmagenta boxes in the growth cabinet under the same conditions.Similarly, plasmids were transformed into Nicotiana using the method ofHorsch et al (R. B. Horsch et al., Science 227, 1229 (Mar. 8, 1985,1985). Maize was transformed with the plasmids by contracting outtransformation services. Similar phenotypes were confirmed in 10independent transgenic lines for 35S::CLE constructs in Arabidopsis.Increases in expression were confirmed in 5 lines per construct byRT-PCR. RT-PCR analysis was carried out using the gene-specific primerslisted in the table. RNA was isolated using Trizol reagent (Invitrogen).cDNA synthesis, following DNase treatment, was performed usingSuperscript III reverse transcriptase (Invitrogen). Expression levels ofCLE41 in wild type were compared to that of 35S::CLE41 by qRT-PCR. Allsamples were measured in technical triplicates on biologicaltriplicates. The qRT-PCR reaction was performed using SYBR GreenJumpStart Taq ReadyMix (Sigma) using an ABI Prism 7000 machine (AppliedBiosystems). PCR conditions were as follows: 50° C. for 2 min, 95° C.for 10 min, and 40 cycles of 95° C. for 15 s and 60° C. for 60 s. Amelting curve was produced at the end of every experiment to ensure thatonly single products were formed. Gene expression was determined using aversion of the comparative threshold cycle (Ct) method. The averageamplification efficiency of each target was determined using LinReg (M.Hardstedt et al., Xenotransplantation 12, 293 (2005)).

Arabidopsis lines which carried 35S::CLE41 35S::PXY and 35S::CLE4235S::PXY were generated by crossing and identified in the F2 population.IRX3::CLE41 35S::PXY and SUC2::CLE41 35S::PXY lines were generated bydirectly transforming plants carrying the 35S::PXY construct withpIRX3::CLE41 or pSUC2::CLE41. SUC2::CLE41 and 35S::CLE41 cell countswere carried out on 10 independent T2's (2 bundles/plant) and 6independent T1's (3 bundles/plant) respectively. 5 week plants wereused. Nicotiana lines carrying 35S::CLE41 35S::PXY were also generatedby crossing.

TABLE 1 Oligonucleotides used in Invention. Seq Id No. Oligo NameSequence (5′-3′) Used for 41 CLE41F CACCATGGCAACATCAAATGAC35S::CLE41 construct 42 CLE41R AAACCAGATGTGCCAACTCA 35S::CLE41 constructand genotyping 43 CLE42F CACCATGAGATCTCCTCACATC 35S::CLE42 construct 44CLE42R TGAATCAAACAAGCAACATAACAA 35S::CLE42 construct and genotyping 45PXY_ORF_f CACCTTAAATCCACCATTGTCA 35S::PXY construct 46 PXY_ORF_rCCAAGATAATGGACGCCAAC 35S::PXY construct 47 SUC2promFtopocaccaacacatgttgccgagtca SUC2::CLE41 overlap PCR entry clone 48SUC2pro/CLE41(1) GTCATTTGATGTTGCCATgaa SUC2::CLE41 overlapatttctttgagagggtttttg PCR entry clone 49 SUC2pro/CLE41(2)caaaaaccctctcaaagaaat SUC2::CLE41 overlap ttcATGGCAACATCAAATGACPCR entry clone 50 CLE41_RTF CCATGACTCGTCATCAGTCC RT-PCR 51 CLE41_RTRTTTGGACCACTAGGAACCTCA RT-PCR 52 CLE42_RTF TCCAAACCCATCAAAGAACC RT-PCR 53CLE42_RTR ATTGGCACCGATCATCTTTC RT-PCR 54 PXY1_RTFAACCTAGCAATATCCTCCTCGAC RT-PCR 55 PXY1_RTR GGTTCCACCGATCTTTTTCC RT-PCR56 ACTS-1 ATGAAGATTAAGGTCGTGGCA RT-PCR control 57 ACTS-2CCGAGTTTGAAGAGGCTAC RT-PCR control 58 qCLE41f TCAAGAGGGTTCTCCTCGAAqRT-PCR 59 qCLE41r TGTGCTAGCCTTTGGACGTA qRT-PCR 60 18s rRNA FCATCAGCTCGCGTTGACTAC qRT-PCR control 61 18s rRNA R GATCCTTCCGCAGGTTCACqRT-PCR control 62 35S promoter F CGCACAATCCCACTATCCTT Genotyping 63pxy-3-r TTACCGTTTGATCCAAGCTTG Genotyping

Histology

Analysis of tobacco, poplar and Arabidopsis vasculature was carried outusing thin transverse sections cut from JB4 resin embedded material asdescribed previously (Pinon et al. 2008). Tissue was fixed in 3%glutaraldehyde or FAA, dehydrated through an ethanol series to 100%ethanol and embedded in JB4 resin (Agar Scientific). Embedded tissue wassectioned at 3 μm and subsequently stained with 0.02% Toluidine Blue.For hand cut sections, tissue was stained with either aqueous 0.02%Toluidine Blue or 0.05M Anniline blue in 100 mM Phosphate buffer, pH7.2.

Stems were analyzed at 8 weeks for Arabidopsis, 50 days for Nicotianaand four weeks after transfer to rooting medium for poplar.

Comparison of Cell Numbers in 35S::CLE41/42 Lines in Arabidopsis.

At the base of 6 week old inflorescence stems, lines over-expressingeither CLE41 or CLE42 had, on average, more undifferentiated cells invascular tissue (105.7 and 89.1, respectively) than those of wild type(58.6). When assaying cell numbers in vascular bundles from multipleinsertion lines, both 35S::CLE41 and 35S::CLE42 plants had more vascularcells, although only in the case of 35S::CLE41 plants was this resultstatistically significant. There was no difference in the number ofdifferentiated vascular cells in either 35S::CLE41 or 35S::CLE42 plantscompared to the wild type (Table 1). We analysed progeny from two of thestronger transformed lines which were also used in subsequent geneticanalysis. Stems from these lines had significantly more cells pervascular bundle (318.7 and 373.7 for 35S::CLE41 and 35S::CLE42,respectively) than wild type (273.7) clearly demonstrating that thesegenes are capable of increasing procambial cell divisions. In the caseof 35S::CLE42 lines there was also a statistically significant increasein the number of differentiated cells.

In order to determine whether these extra procambial cells would remainundifferentiated or would differentiate into xylem and phloem, we lookedat the base of plant stems at senescence. In all genotypes the vastmajority of vascular cells in the stem were fully differentiated (FIG.5), including areas in 35S::CLE41/42 where large numbers ofundifferentiated cells were present at earlier stages of development.Therefore, early on 35S::CLE41/42 plants have more undifferentiatedcells but these ultimately become differentiated in inflorescence stems.

Mean vascular cell number from 19 independent transgenic lines.35S::CLE42 Col (n = 10) 35S::CLE41 (n = 9) (n = 10) Total Cells 311.6 ±15.6 373.2^(φ) ± 24.3  341.8 ± 19.1 Undifferentiated Cells 58.6 ± 4.4105.7* ± 9.8  89.0* ± 7.2  Differentiated Cells   253 ± 12.6 267.5 ±15.2 252.8 ± 14.4 (Xylem and Phloem) *Significantly different from Col P< 0.001. ^(φ)Significantly different from Col p < 0.05. ± Standarderror.Over-Expression of CLE41 and CLE42 in Conjunction with PXY FurtherEnhances Effects on Secondary Growth

We addressed the consequences of expressing PXY and CLE41 by using a35S::PXY construct in a 35S::CLE41/42 background. The stems of35S::CLE41/42 35S::PXY plants were characterised by dramatic increasesin cell number in both the vascular bundle and in the interfascicularregion such that a continuous ring of additional tissue within the stem.New cells were generated between the xylem and phloem in vascularbundles and also outside the interfascicular cells making the phenotypecharacteristic of dramatically increased secondary growth (FIG. 8).These results provide strong genetic evidence that CLE41/42 and PXY aresufficient for induction of vascular cell division within the procambiumand elsewhere. Interestingly, the majority of increased cell divisionsoccurring when both CLE41/42 and PXY are over-expressed were relativelyordered, although aberrant cells divisions are still present. We madelines harbouring both IRX3::CLE41 and 35S::PXY constructs. We found thatvascular organisation was disrupted in 35S::PXY IRX3::CLE41 plants (FIG.25?), but increased secondary growth was also observed. 35S::PXYSUC2::CLE41 plants also demonstrated enhanced secondary growth (FIG.22), but in contrast to 35S::PXY IRX3::CLE41, vascular tissue was highlyordered.

An additional phenotype was observed in the leaves of 35S::CLE41/4235S::PXY plants. In Col, 35S::CLE41/42 (FIG. 9) and 35S::PXY, leaveshave a single midvein, however, in a minority of 35S::CLE42 35S::PXYplants the leaves appeared to exhibit increased vascular development.This additional vascular tissue develops together with the associatedlamina suggesting development of ectopic vascular tissue.

Identification of CLE and PXY Homologues

Identification of Populus Trichocarpa CLE family was carried out bysubjecting CLE41/42 to a WU-BLAST search against green plant GB genomic(DNA) datatsets using TBLASTN: AA query to NTdb parameters on the TAIRwebsite (www.arabidopsis.org). All Popolus trichocarpa hits (genomicregion) with probability value (P) less than 1 were selected. These hitswere subsequently on the Populus gene map(http://www.ncbi.nlm.mih.gov/projects/mapview/map_search.cgi?taxid=3694).±1 kb from the WU-BLAST hit region was then analyzed with the NCBI ORFfinder (http://www.ncbi.nlm.nih.gov/projects/gorf/) and all codingregions containing similar 12 AA sequences to the output CLE sequencewere examined. All putative proteins were aligned using the ClustalWalgorithm using default settings.

The PXY homolog in Oryza Sativa was identified by locating PXY(At5g61480) in the homology tree from Shiu et al. (S.-H. Shiu et al.,Plant Cell 16, 1220 (May 1, 2004, 2004)). The putative homolog wasOsi056321.1 (Oryza Sativa Indica). This sequence was then subjected to aBLASTP protein search against O. sativa (japonica cultivar-group)Non-RefSeq protein. The top hit was EAZ41508.1: hypothetical proteinOsJ_(—)024991 and was confirmed as being the PXY orthologue byperforming a BLASTP OsJ_(—)024991 against Non-RefSeq protein database,Arabidopsis Thaliana, NCBI.

TABLE 2 ID (representative Protein/Gene CLE Representative SynonymsOrganism seq) Annotation 41 AT_GEN_At3g24770.1 CLE41 ArabidopsisAT_GEN_At3g24770.1 At3g24770.1 thaliana 68416.m03109 CLE41, putativeCLAVATA/ESR- Related 41 (CLE41) ATEST_TC255991_+1 TC255991 AT- TA130113702 Putative TA_TA13011_3702_+1 CLE41 protein related cluster 42ATEST_NP1098871_+1 CLE42 Arabidopsis ATEST_NP1098871_+1 NP1098871putative thaliana CLAVATA3/ESR-related 42 precursor [Arabidopsisthaliana] 44 AT_GEN_At4g13195.1 CLE44 Arabidopsis AT_GEN_At4g13195.1At4g13195.1 thaliana 68417.m02052 expressed protein ATEST_TC275167_+3TC275167 GB|AAO11557.1|27363276| BT002641 At4g13194/At4g13194{Arabidopsis thaliana;}, complete 51 GMEST_TC171126_+3 GlycineGMEST_TC171126_+3 TC171126 similar to max PIR|S61040|S61040 probablemembrane protein YDL172c - yeast (Saccharomyces cerevisiae), partial(11%) GM- TA36215 3847 TA_TA36215_3847_−3 53 GMEST_TC162846_+3 GlycineGMEST_TC162846_+3 TC162846 homologue to max GP|21618281|gb|AAM67331.1unknown {Arabidopsis thaliana}, partial (27%) GMEST_TC162847_+1 TC162847homologue to GP|21618281|gb|AAM67331.1 unknown {Arabidopsis thaliana},partial (19%) GM-TA_BQ627547_−1 BQ627547 Hypothetical protein CBG22664related cluster GM- TA14900 3847 TA_TA14900_3847_+1 GM- TA8421 3847SPBC215.13 TA_TA8421_3847_+3 protein related cluster 60GMEST_BE658554_−3 Glycine GMEST_BE658554_−3 BE658554 homologue to maxPIR|H72173|H7217 D5L protein - variola minor virus (strain Garcia-1966),partial (23%) GM-TA_BE658554_−3 BE658554 61 GMEST_BM085374_+2 GlycineGMEST_BM085374_+2 BM085374 homologue to max GP|16945432|emb related toGLUCAN 1 3-BETA-GLUCOSIDASE PRECURSOR protein {Neurospora crassa},partial (1%) GM- TA39380 3847 TA_TA39380_3847_+1 62 GMEST_TC171467_+2Glycine GMEST_TC171467_+2 TC171467 max GM- TA31733 3847TA_TA31733_3847_+2 63 GMEST_BU763224_+1 Glycine GMEST_BU763224_+1BU763224 similar to max GP|21592472|gb|CLE gene family putative{Arabidopsis thaliana}, partial (15%) GM- BU763224 TA_BU763224_+1 64MT_GEN_IMGA|AC137080_19.1 Medicago MT_GEN_IMGA|AC137080_19.1IMGA|AC137080_19.1 truncatula AC137080.13 104569- 102347 E EGN_Mt04120920041210 hypothetical protein MT_GEN_IMGA|AC147499_5.1 IMGA|AC147499_5.1AC147499.5 26650-24428 E EGN_Mt041209 20041210 hypothetical proteinMTEST_BI311733_+1 BI311733 MT- BI311733 TA_BI311733_+1 90OS_GEN_Os02g56490.1 Oryza OS_GEN_Os02g56490.1 Os02g56490.1|11972.m33318|sativa protein expressed protein OSEST_TC278386_+3 TC278386 Oryza sativa(japonica cultivar- group) cDNA clone: J033127D10, full insert sequenceOS- TA21276 4530 TA_TA21276_4530_+2 Hypothetical protein OJ1520 C09.33related cluster 116 PT_GEN_63277 Populus PT_GEN_63277 jgi|Poptr1|63277|trichocarpa fgenesh1_pg.C_LG_I000629 119 PT_GEN_569594 PopulusPT_GEN_569594 jgi|Poptr1|569594|eugene3.00120247 trichocarpa 148ZMEST_DR801316_+1 Zea mays ZMEST_DR801316_+1 DR801316 ZM-TA_DR801316_−1DR801316 Hypothetical protein P0617C02.125 related cluster 149ZMEST_BM350390_−2 Zea mays ZMEST_BM350390_−2 BM350390 similar toUP|Q4NZF2 9DELT (Q4NZF2) PE-PGRS family protein, partial (4%) ZM-BM350390 PE-PGRS TA_BM350390_+2 family protein related cluster 166STEST_TC114822_+3 Solanum STEST_TC114822_+3 TC114822 TIGR tuberosumAth1|At4g13195.1 68417.m02052 expressed protein, partial (12%) ST-TA8910 4113 TA_TA8910_4113_+3 Hypothetical protein MTH423 relatedcluster 167 STEST_BF187584_+1 Solanum STEST_BF187584_+1 BF187584tuberosum ST- TA17128 4113 Cluster TA_TA17128_4113_+2 related toUPI0000517AA3 168 BN- Brassica BN- CX187708 F20P5.29 TA_CX187708_+3napus TA_CX187708_+3 protein related cluster 169 PV- Phaseolus PV-CV532906 Hypothetical TA_CV532906_+1 vulagaris TA_CV532906_+1 proteinrelated cluster 172 STEST_TC129811_+3 Solanum STEST_TC129811_+3 TC129811similar to tuberosum TIGR Ath1|At4g13195.1 68417.m02052 expressedprotein, partial (12%) ST- TA11604 4113 TA_TA11604_4113_+3 Hypotheticalprotein related cluster 173 STEST_CV500295_+3 Solanum STEST_CV500295_+3CV500295 tuberosum ST- TA19709 4113 TA_TA19709_4113_+3 Hypotheticalprotein related cluster

Nicotiana Over Expressing CLE41/42 and PXY

In order to observe the phenotypic differences between the transgenicplants and wild type in Nicotiana, 35S::PXY, 35S::CLE41, 35S::CLE42 andwild type plants were grown in individual pots and places in the growthcabinet under same conditions. The height (from soil surface to theplant top in cM), hypocotyl width and stem width (diameter in mm) weremeasured when plants were 50 days old. The results of mean, standarderror (SE), standard deviation (STD), minimum (Min) and maximum (Max)value were summarised in tables 3, 4, and 5. Single ANOVA betweentransgenic lines and wild type have been analysed and the P-value weregiven in the tables as well.

In table 3, the results show that the height of transgenic lines arehighly significant difference between wild type (P<0.001****), the meansof 35S::CLE41 and 35S::CLE42 are similar. 35S::CLE41 and 35S::CLE42 arealso 20 cM and 12 cM shorter than wild type and 35S::PXY respectively.The results are consistent with the phenotypes being induced by overexpression of CLE41 and CLE42.

TABLE 3 Height of Nicotiana at 50 days (cM) Name of plant N Mean ±SE STDMin Max P-value 35S::PXY 10 26.70 1.10 3.49 18 30.5 <0.001****35S::CLE41 10 14.40 1.37 4.34 5.5 20 <0.001**** 35S::CLE42 8 15.98 3.329.38 6 29 <0.001**** Wild type 10 35.35 1.20 3.79 27 39

Table 4, shows that Hypocotyl width is not significantly differentbetween 35S::PXY and wild type, however, there is a highly significantdifference between 35S::CLE41 or 35S::CLE42 and Wild type (P<0.0001***).The means of 35S::CLE41 and 35S::CLE42 are about 2.3 mm thicker thanwild type. The maximum hypocotyls width is 10.29 mm in 35S::CLE41compared to 7.1 mm in wild type, there is a 3.19 mm difference,demonstrating that overexpression of CLE41 and CLE42 increases hypocotylwidth.

TABLE 4 Hypocotyl width at 50 days (mm) (Nicotiana) Name of plant N Mean±SE STD Min Max P-value 35S::PXY 10 6.48 0.20 0.63 5.2 7.41 >0.0535S::CLE41 10 8.71 0.30 0.96 6.87 10.29 <0.0001*** 35S::CLE42 8 8.930.35 0.99 7.1 10.06 <0.0001*** Wild type 10 6.34 0.14 0.45 5.3 7.1

In table 5, the results show that there is no significant differencebetween 35S::PXY and wild type stem width, however, there is highlysignificant difference between 35S::CLE41 or 35S::CLE42 and Wild type(P<0.0001****). The maximum stem width is 8.62 mm in 35S::CLE41 comparedwith 5.92 mm in wild type, there is 2.7 mm different. The results shownthat 35S::PXY did not affect the stem width, while the overexpression ofCLE41 and CLE42 made the stem thicker than wild type.

TABLE 5 Stem width at 50 days (mm) (Nicotiana) Name of plant N Mean ±SESTD Min Max P-Value 35S::PXY 10 5.23 0.22 0.68 4.3 6.67 >0.05 35S::CLE4110 7.06 0.33 1.03 5.24 8.62 <0.001**** 35S::CLE42 8 6.89 0.40 1.13 4.968.06 <0.001**** Wild type 10 5.49 0.11 0.36 4.8 5.92

In summary, over expression of PXY results in a significant change tothe plant height compared to the wild type. However, over expression ofCLE41 and CLE42 significantly alter the plants phenotype in terms ofheight, hypocotyl width and stem width.

2. Nicotiana Images

In order to observe the phenotypic changes between the transgenic linesand wild type, the hypocotyl sections of transgenic lines 35S::PXY,35S::CLE41, 35S::CLE42, 35S::CLE41 35S::PXY and wild type were cut whenthe plants were 50 days old. The images of whole plants and hypocotylcross sections in FIGS. 19 and 20 illustrate the phenotypic differencesbetween the lines. FIG. 19, shows that the over expression CLE41 resultsin a dwarf phenotype as documented in the table 4. The photographsdemonstrate that this defect is much less when both CLE41 and PXY areover expressed.

In FIG. 20, cross sections of hypocotyls of plants demonstrates thatplants over expressing PXY and CLE41 have thicker hypocotyls.

3. Histological Analysis of Nicotiana Hypocotyl Sections.

FIG. 21, wild type (A), 35S::CLE41 (B), 35S::CLE42 (C), 35S::PXY (D),35S::CLE41 35S::PXY(E) Organisation is lost in plants over expressingCLE41/CLE42. Organisation is restored in plants over expressing bothCLE41 and PXY.

A further experiment illustrates differences between single overexpression of PXY, CLE41 and both PXY and CLE41. 10 plants of genotypes35S::PXY, 35S::CLE41, 35S::CLE41 35S::PXY and wild type were planted inan individual pots and grown in a growth cabinet in identicalconditions. The height (from soil surface to the plant top in cm),hypocotyl width and stem width (diameter in mm) were measured whenplants were 42 days old. The results of mean, standard error (SE),standard deviation (STD), minimum (Min) and maximum (Max) value arepresented in tables 6, 7, and 8. Single ANOVA between transgenic linesand wild type have been analysed and the P-value were given in thetables.

TABLE 6 Height at 42 days (cM) Table 6. The height of 35S::CLE41 plantsare significantly smaller than wild type (P < 0.001****), however, thereis no difference between 35S::CLE41 35S::PXY and wild type in height.Name N Mean SE STD Min Max P-value 35S::CLE41 10 9.6 0.42 1.34 7 11.5 P< 0.0001**** 35S::PXY 10 23.8 1.34 4.26 16 31.5 P < 0.01**   35S::CLE4110 16.95 1.45 4.58 12 23 P > 0.05    35S::PXY Wild type 10 19.12 0.621.98 16.5 22

TABLE 7 Nicotiana Hypocotyls at 42 days (mm) Table 7. Hypocotyl widthwas significantly larger in 35S::CLE41 and 35S::CLE41 35S::PXY comparedto Wild type (P < 0.0001***). The mean of 35S::CLE41 is about 2.4 mmthicker than wildtype. The mean of 35S::CLE41 35S::PXY is 2.7 mm thickerthan wild type. Name N Mean SE STD Min Max P-value 35S::CLE41 10 9.810.35 1.11 8.27 11.58 P < 0.0001**** 35S::PXY 10 8.31 0.39 1.25 6.47 9.86P > 0.05    35S::CLE41 10 10.22 0.39 1.24 8.19 11.58 P < 0.0001****35S::PXY Wild type 10 7.45 0.27 0.87 5.99 8.87

TABLE 8 Stem width at 42 days (mm) Table 8. There is highly significantdifference between 35S::CLE41 and 35S::CLE41 35S::PXY compared to wildtype (P < 0.0001****). There is also a significant difference between35S:PXY and Wild type. The maximum stem width is 9.43 mm in 35S::CLE41compare 6.74 mm in wild type, a difference of 2.7 mm. The resultsdemonstrate that overexpression of both CLE41 and PXY increase stemthickness compared to wild type. Name N Mean SE STD Min Max P-value35S::CLE41 10 8.49 0.22 0.70 7.33 9.43 P < 0.0001**** 35S::PXY 10 6.810.37 1.19 4.58 8.27 P < 0.05*    35S::CLE41 10 8.18 0.36 1.14 6.86 9.69P < 0.0001**** 35S::PXY Wild type 10 5.88 0.16 0.53 5.14 6.74Poplar Harbouring 35S::CLE41 or SUC2::CLE41 Constructs Generate MoreVascular Tissue than Wild Type.

Poplar transformed with 35S::CLE41 or SUC2::CLE41 were in JB4 sectionswere found to have more xylem tissue (see brackets in FIG. 25)demonstrating increases in vascular tissue.

1. Use of a polypeptide selected from the group consisting of: i) aCLE41 polypeptide; ii) a CLE42 polypeptide; iii) a polypeptidecomprising an amino acid sequence that is at least 70% identical to theamino acid sequence of amino acids 124 to 137 of the consensus sequenceof FIG. 10; iv) a polypeptide comprising an amino acid sequence that isat least 70% identical to the amino acid sequence of CLE41 of FIG. 13Aor CLE42 of FIG. 14A; v) a polypeptide encoded by a nucleic acidmolecule that is at least 70% identical to the nucleotide sequence ofCLE41 of FIG. 13B or CLE42 of FIG. 14B; or a nucleic acid moleculeselected from the group consisting of vi) a nucleic acid molecule thatencodes a CLE41 polypeptide; vii) a nucleic acid molecule that encodes aCLE42 polypeptide; viii) a nucleic acid molecule that encodes apolypeptide comprising an amino acid sequence that is at least 70%identical to the amino acid sequence of amino acids 124 to 137 of theconsensus sequence of FIG. 10; ix) a nucleic acid molecule which is atleast 70% identical to the nucleotide sequence of CLE41 of FIG. 13B orCLE42 of FIG. 14B; x) a nucleic acid molecule that is hybridizes understringent conditions to the nucleotide sequence vi) or vii) in themanipulation of plant growth and/or structure.
 2. (canceled)
 3. Useaccording to claim 1 in combination with a nucleic acid moleculeselected from the group consisting of: i) a nucleic acid molecule thatencodes a CLE41 receptor; ii) a nucleic acid molecule that encodes aCLE42 receptor; iii) a nucleic acid molecule that encodes a polypeptidecomprising an amino acid sequence that is at least 70% identical to theconsensus sequence of FIG. 12, or a functional equivalent thereof; iv) anucleic acid molecule that is at least 70% identical to the nucleotidesequence of FIG. 15B or 15C; v) a nucleic acid molecule that is at least70% identical to a nucleic acid molecule of i) or ii); vi) a nucleicacid molecule that is hybridizes under stringent conditions to thenucleotide sequence i) or ii); or a polypeptide selected from the groupconsisting of vii) a CLE41 receptor; viii) a CLE42 receptor; ix) apolypeptide comprising an amino acid sequence that is at least 70%identical to the consensus sequence of FIG. 12, or a functionalequivalent thereof x) a polypeptide comprising an amino acid sequencethat is at least 70% identical to the PXY sequence of FIG. 15A; xi) apolypeptide sequence comprising an amino acid sequence which is at least70% identical to a sequence encoding vii) or viii); xii) a polypeptidecomprising an amino acid sequence encoded by a nucleic acid moleculethat is at least 70% identical to the PXY nucleotide sequence of FIG.15B or 15C.
 4. (canceled)
 5. Use according to claim 3, wherein saidCLE41 or CLE42 receptor is PXY or a functional equivalent thereof.
 6. Amethod of manipulating the growth and/or structure of a plant,comprising modulating the level of CLE41 and/or CLE42 or a functionalequivalent thereof, in the plant, wherein the levels of CLE41 and/orCLE42 are modulated by introducing into a cell of the plant: i) a CLE41polypeptide; ii) a CLE42 polypeptide; iii) a polypeptide comprising anamino acid sequence that is at least 70% identical to the amino acidsequence of amino acids 124 to 137 of the consensus sequence of FIG. 10;iv) a polypeptide comprising an amino acid sequence that is at least 70%identical to the amino acid sequence of CLE41 of FIG. 13A or CLE42 ofFIG. 14A; v) a polypeptide encoded by a nucleic acid molecule that is atleast 70% identical to the nucleotide sequence of CLE41 of FIG. 13B orCLE42 of FIG. 14B, or vi) a nucleic acid molecule that encodes a CLE41polypeptide; vii) a nucleic acid molecule that encodes a CLE42polypeptide; viii) a nucleic acid molecule that encodes a polypeptidecomprising an amino acid sequence that is at least 70% identical to theamino acid sequence of amino acids 124 to 137 of the consensus sequenceof FIG. 10; ix) a nucleic acid molecule which is at least 70% identicalto the nucleotide sequence of CLE41 of FIG. 13B or CLE42 of FIG. 14B; x)a nucleic acid molecule that is hybridizes under stringent conditions tothe nucleotide sequence vi) or vii).
 7. (canceled)
 8. (canceled)
 9. Amethod according to claim 6, wherein the levels of levels of CLE41and/or CLE42 or a functional equivalent thereof are upregulated.
 10. Amethod according to claim 6, further comprising introducing into a cellof the plant: i) a nucleic acid molecule that encodes a CLE41 receptor;ii) a nucleic acid molecule that encodes a CLE42 receptor; iii) anucleic acid molecule that encodes a polypeptide comprising an aminoacid sequence that is at least 70% identical to the consensus sequenceof FIG. 12, or a functional equivalent thereof; iv) a nucleic acidmolecule that is at least 70% identical to the nucleotide sequence ofFIG. 15B or 15C; v) a nucleic acid molecule that is at least 70%identical to a nucleic acid molecule of i) or ii); vi) a nucleic acidmolecule that is hybridizes under stringent conditions to the nucleotidesequence i) or ii), or a polypeptide selected from the group consistingof vii) a CLE41 receptor; viii) a CLE42 receptor; ix) a polypeptidecomprising an amino acid sequence that is at least 70% identical to theconsensus sequence of FIG. 12, or a functional equivalent thereof; x) apolypeptide comprising an amino acid sequence that is at least 70%identical to the PXY sequence of FIG. 15A; xi) a polypeptide sequencecomprising an amino acid sequence which is at least 70% identical to asequence encoding vii) or viii); xii) a polypeptide comprising an aminoacid sequence encoded by a nucleic acid molecule that is at least 70%identical to the PXY nucleotide sequence of FIG. 15B or 15C. 11.(canceled)
 12. A method according to claim 10, wherein said CLE41 and/orCLE42 receptor is PXY or a functional equivalent thereof.
 13. A plantcell manipulated to express: i) a CLE41 polypeptide; ii) a CLE42polypeptide; iii) a polypeptide comprising an amino acid sequence thatis at least 70% identical to the amino acid sequence of amino acids 124to 137 of the consensus sequence of FIG. 10; iv) a polypeptidecomprising an amino acid sequence that is at least 70% identical to theamino acid sequence of CLE41 of FIG. 13A or CLE42 of FIG. 14A; v) apolypeptide encoded by a nucleic acid molecule that is at least 70%identical to the nucleotide sequence of CLE41 of FIG. 13B or CLE42 ofFIG. 14B; or a nucleic acid molecule selected from the group consistingof a nucleic acid molecule that encodes a CLE41 polypeptide; vii) anucleic acid molecule that encodes a CLE42 polypeptide; viii) a nucleicacid molecule that encodes a polypeptide comprising an amino acidsequence that is at least 70% identical to the amino acid sequence ofamino acids 124 to 137 of the consensus sequence of FIG. 10; ix) anucleic acid molecule which is at least 70% identical to the nucleotidesequence of CLE41 of FIG. 13B or CLE42 of FIG. 14B; x) a nucleic acidmolecule that is hybridizes under stringent conditions to the nucleotidesequence vii) or viii); wherein the expression is optionally incombination with expression of a receptor for CLE41 and/or CLE42. 14.(canceled)
 15. A plant cell according to claim 13 further manipulated toexpress a nucleic acid molecule selected from the group consisting of:i) a nucleic acid molecule that encodes a CLE41 receptor; ii) a nucleicacid molecule that encodes a CLE42 receptor; iii) a nucleic acidmolecule that encodes a polypeptide comprising an amino acid sequencethat is at least 70% identical to the consensus sequence of FIG. 12, ora functional equivalent thereof; iv) a nucleic acid molecule that is atleast 70% identical to the nucleotide sequence of FIG. 15B or 15C; v) anucleic acid molecule that is at least 70% identical to a nucleic acidmolecule of i) or ii); vi) a nucleic acid molecule that is hybridizesunder stringent conditions to the nucleotide sequence i) or ii) or apolypeptide selected from the group consisting of vii) a CLE41 receptor;viii) a CLE42 receptor; ix) a polypeptide comprising an amino acidsequence that is at least 70% identical to the consensus sequence ofFIG. 12, or a functional equivalent thereof x) a polypeptide comprisingan amino acid sequence that is at least 70% identical to the PXYsequence of FIG. 15A; xi) a polypeptide sequence comprising an aminoacid sequence which is at least 70% identical to a sequence encodingvii) or viii); xii) a polypeptide comprising an amino acid sequenceencoded by a nucleic acid molecule that is at least 70% identical to thePXY nucleotide sequence of FIG. 15B or 15C.
 16. (canceled)
 17. A plantcomprising a cell according to claim
 13. 18. (canceled)
 19. A method ofmanipulating the growth and/or structure of a plant, comprising thesteps of: i) providing a cell claim 13; ii) regenerating said cell intoa plant; and optionally iii) monitoring the levels of CLE41 and/or CLE42or a receptor thereof, and or PXY or functional equivalents thereof insaid regenerated plant.
 20. An expression construct comprising a firstnucleic acid sequence selected from the group consisting of: i) anucleic acid molecule that encodes a CLE41 polypeptide; ii) a nucleicacid molecule that encodes a CLE42 polypeptide; iii) a nucleic acidmolecule that encodes a polypeptide comprising an amino acid sequencethat is at least 70% identical to the amino acid sequence of amino acids124 to 137 of the consensus sequence of FIG. 10; iv) a nucleic acidmolecule which is at least 70% identical to the nucleotide sequence ofCLE41 of FIG. 13B or CLE42 of FIG. 14B; v) a nucleic acid molecule thatis hybridizes under stringent conditions to the nucleotide sequence i)or ii); and optionally a second nucleotide sequence encoding aregulatory sequence capable of expressing the first nucleotide sequencespecifically in or adjacent to the vascular tissue of a plant.
 21. Anexpression cassette according to claim 20 further comprising: i) anucleic acid molecule that encodes a CLE41 receptor; ii) a nucleic acidmolecule that encodes a CLE42 receptor; iii) a nucleic acid moleculethat encodes a polypeptide comprising an amino acid sequence that is atleast 70% identical to the consensus sequence of FIG. 12, or afunctional equivalent thereof; iv) a nucleic acid molecule that is atleast 70% identical to the nucleotide sequence of FIG. 15B or 15C; v) anucleic acid molecule that is at least 70% identical to a nucleic acidmolecule of i) or ii); vi) a nucleic acid molecule that is hybridizesunder stringent conditions to the nucleotide sequence i) or ii).
 22. Ahost cell or organism comprising an expression construct of claim 21.23. A transgenic plant seed comprising a cell according to claim
 13. 24.(canceled)
 25. A method of producing a plant-derived product comprising:i) manipulating the growth and/or structure of a plant producedaccording to claim 6; ii) growing the plant until it reaches apre-determined lateral size; and optionally iii) harvesting the plantderived product of the plant.
 26. A method of altering the mechanicalproperties of a plant or plant derived product comprising: i)manipulating the growth and/or structure of a plant according to claim6; ii) growing the plant until it reaches a pre-determined size; andoptionally iii) harvesting a plant derived product of the plant.
 27. Aplant derived product produced by a method according to claim 25.